Trehalose phosphorylase, its preparation and uses

ABSTRACT

A thermostable trehalose phosphorylase which is obtainable from microorganisms of the genus Thermoanaerobium and which hydrolyzes trehalose in the presence of an inorganic phosphoric acid to form D-glucose and β-D-glucose-1-phosphoric acid. The trehalose phosphorylase can be also prepared by recombinant DNA technology. When the enzyme is allowed to contact with β-D-glucose-1-phosphoric acid as a saccharide donor in the presence of other saccharides, glucosyl-transferred saccharides including glucosyl-D-galactoside, which are conventionally known but scarcely obtainable, can be produced on an industrial-scale and in a relatively-low cost.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of copending parent application Ser. No. 08/966,389,filed Nov. 7, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel trehalose phosphorylase, itspreparation and uses, more particularly, to a novel trehalosephosphorylase which hydrolyzes trehalose in the presence of an inorganicphosphoric acid and/or its salt (hereinafter abbreviated as "inorganicphosphoric acid" throughout the present specification, if not anyinconvenience will arise) to form D-glucose and β-D-glucose-1-phosphoricacid and/or its salt (hereinafter abbreviated as"β-D-glucose-1-phosphoric acid" throughout the present specification, ifnot any inconvenience will arise), and which, in reverse, formstrehalose and inorganic phosphoric acid from β-D-glucose-1-phosphoricacid and D-glucose, and to the processes of the trehalose phosphorylase,saccharide compositions containing glucosyl-transferred saccharidesproduced by using the trehalose phosphorylase, and compositionscontaining the saccharide compositions.

2. Description of the Prior Art

Recently, oligosaccharides such as maltose and trehalose and functionsthereof have become to be highlighted, and have been studied on theirunique and different processes in view of various aspects. It is knownthat phosphorylases such as maltose, trehalose, sucrose, trehalose, andcellobiose phosphorylases can be used as methods for producing the aboveoligosaccharides.

L. R. Marechal et al reported that in "The Journal of BiologicalChemistry", Vol.247, No.10, pp.3,223-3,228 (1972), Euglena gracilisproduces trehalose phosphorylase intracellularly; and S. Murao et alreported that in "Agriculture and Biological Chemistry", Vol.49, No.7,pp.2,113-2,118 (1985), the properties of the enzyme. K. Aisaka et aldisclosed in Japanese Patent Kokai No.59,584/95 a bacterial trehalosephosphorylase which is produced by a microorganism of the speciesCatellatospora ferruginea and one of the species Kineosporia aurantiaca.H. Kizawa et al reported that in "Bioscience, Biotechnology andBiochemistry", Vol.59, No.10, pp.1,908-1,912 (1995), a microorganism ofthe species Micrococcus varians produces such an enzyme, and M. Yoshidaet al reported that in "Oyo-Toshitsu-Kagaku", Vol.42, No.1, pp.19-25(1995), a microorganism of the species Plesiomonas sp. SH-35 producestrehalose phosphorylase. Among these trehalose phosphorylases, theenzymes from the microorganisms of the species Micrococcus varians,Euglena gracilis, and Plesiomaonas sp. have lower thermal stabilities ofless than 30, 40 and 45° C., respectively, resulting both in arelatively-low reaction efficiency on an industrial scale production andin a bacterial contamination during an enzymatic reaction.

SUMMARY OF THE INVENTION

The present invention provides a novel trehalose phosphorylase with anindustrially-advantageous thermostability, process thereof, saccharidecompositions containing the glucosyl-transferred saccharides prepared byusing the enzyme, and uses thereof.

To solve the above object and to obtain an unknown trehalosephosphorylase with a satisfactory thermostability, the present inventorswidely screened microorganisms which produce such an enzyme. As aresult, they found that Thermoanaerobium brockii, ATCC 35047, belongingto the genus Thermoanaerobium, produces a novel trehalose phosphorylaseand established the preparation. They also established a saccharidecomposition containing glucosyl-transferred saccharides produced bycontacting the enzyme with β-D-glucose-1-phosphoric acid as a saccharidedonor in the presence of saccharides, and compositions containing thesaccharide composition. Thus, they accomplished this invention.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 shows the influence of temperatures on the activity of trehalosephosphorylase according to the present invention.

FIG. 2 shows the influence of pHs on the activity of trehalosephosphorylase according to the present invention.

FIG. 3 shows the influence of temperatures on the stability of trehalosephosphorylase according to the present invention.

FIG. 4 shows the influence of pHs on the stability of trehalosephosphorylase according to the present invention.

FIG. 5 is a restriction map of the present recombinant DNA. In thefigure, an arrow shows a DNA encoding the present trehalosephosphorylase.

DETAILED DESCRIPTION OF THE INVENTION

The trehalose phosphorylase according to the present invention includesenzymes which are obtainable from microorganisms of the genusThermoanaerobium and which hydrolyze trehalose in the presence of aninorganic phosphoric acid to form D-glucose and β-D-glucose-1-phosphoricacid. The trehalose phosphorylase may have the following actions andphysicochemical properties:

(1) Action

(a) Hydrolyzing trehalose in the presence of an inorganic phosphoricacid to form D-glucose and β-D-glucose-1-phosphoric acid;

(b) Forming trehalose and an inorganic phosphoric acid from D-glucoseand β-D-glucose-1-phosphoric acid, and catalyzing the transfer reactionof glucosyl group to other saccharides using β-D-glucose-1-phosphoricacid as a saccharide donor;

(2) Molecular weight

88,000±5,000 daltons on SDS-PAGE;

(3) Isoelectric point

pI 5.4±0.5 on electrophoresis using ampholyte;

(4) Optimum temperature

About 70° C. when incubated at pH 7.0 for 30 min;

(5) Optimum pH

About 7.0-7.5 when incubated at.60° C. for 30 min;

(6) Thermal stability

Stable up to a temperature of about 60° C. when incubated at pH 7.0 forone hour;

(7) pH Stability

Stable at pHs of about 6.0-9.0 when incubated at 4° C. for 24 hours;

(8) Activation and stabilization

Activated by one mM dithiothreitol; and

(9) Activity inhibition

Inhibited by one mM Cu⁺⁺, Pb⁺⁺, Zn⁺⁺, Hg⁺⁺, Mg⁺⁺, or Mn⁺⁺.

The trehalose phosphorylase may have the amino acid sequences of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 as a partial amino acid sequence, andas a whole, may have the amino acid sequence of SEQ ID NO:4. In thisart, if there exists an isolated microorganism which produces a desiredprotein, functional equivalents of the protein can be easily obtained bytreating the microorganism with an appropriate mutagen, or if thereexists a DNA encoding a desired protein, functional equivalents of theprotein can be easily obtained by applying recombinant DNA technology ingeneral to the DNA. The present trehalose phosphorylase, of course,includes such functional equivalents. The wording "functionalequivalents" means proteins which still have substantially the sameactivity of the trehalose phosphorylase, and have the amino acidsequence of the enzyme where one or more amino acids are replaced withdifferent ones, one or more amino acids are either added to the N-and/or C-termini or inserted into the internal amino acid sequence, oneor more amino acids in the N- and/or C-terminal regions are deleted, andone or more amino acids in the internal amino acid sequence are deleted.The present trehalose phosphorylase as mentioned above can besatisfactorily obtained by separating it from natural resources such ascultures of microorganisms which produce the enzyme, mutants thereofobtained by treating with mutagens, and artificially synthesizing theenzyme by applying recombinant DNA and peptide-synthesizingtechnologies.

The DNA according to the present invention includes all the above DNAswhich encode the present trehalose phosphorylase. Examples of such DNAsare those which contain the amino acid sequence of SEQ ID NO:4 andencode the nucleotide sequence of SEQ ID NO:5, and functionalequivalents thereof. The wording "functional equivalents" means DNAswhich encode proteins, substantially have the same activity as thetrehalose phosphorylase, and have the nucleotide sequence of SEQ ID NO:5where one or more bases are replaced with different ones. In addition tothe above DNAs, the present DNA includes another DNAs where the 5'-and/or 3'-termini are linked to one or more DNAs other than the aboveDNAs, such as start codon initiators, stop codons, Shine-Dalgarnosequence, nucleotide sequences encoding signal peptides, recognitionsequences by appropriate restriction enzymes, promoters, enhancers, andterminators.

Resources and preparations of these DNAs are not specifically restrictedin the present invention. For example, microorganisms of the genusThermoanaeroblum including Thermoanaerobium brockii, ATCC 35047, asnatural resources of the DNAs, can be mentioned. DNAs containing thepresent DNA can be obtained by collecting DNA fractions from the celldebris of cultured microorganisms. The collected DNAs per se can be usedin the present invention, and they can be prepared into an extremelyfavorable recombinant DNA by introducing a fragment containing thepresent DNA into a self-replicable vector. The recombinant DNA can begenerally obtained by applying the recombinant DNA technology in generalto the above DNA to obtain a gene library, and applying a selectionmethod such as hybridization method for selecting the desiredrecombinant DNA from the gene library based on the nucleotide sequence,which encodes the present trehalose phosphorylase, such as SEQ ID NO:5.The recombinant DNA thus obtained can be amplified when culturedtransformants obtained by introducing into appropriate hosts such asmicroorganisms of the genus Escherichia, followed by applying thealkali-SDS method in general to the cultures to easily obtain thepresent DNA in a desired amount. The present DNA can be easily obtainedby applying the PCR method in a conventional manner using as a templatethe disrupted cells of the above microorganisms or the DNA collectedfrom the cells, and using as a primer a DNA chemically synthesized basedon SEQ ID NO:5, or by chemically synthesizing a DNA containing SEQ IDNO:5. The above functional equivalents of the present DNA can beobtained, for example, by applying the site-directed mutagenesis to theabove recombinant DNA, or applying the PCR method using both therecombinant DNA as a template and a chemically synthesized DNAcontaining a nucleotide sequence, which was converted into the desirednucleotide sequence, as a primer.

The present DNA includes those in the form of a recombinant DNA which isintroduced into a self-replicable vector. As described above, theserecombinant DNAs are extremely useful in preparing the present DNA, andare also useful in producing the present trehalose phosphorylase. Oncethe desired DNA is obtained as described above, those recombinant DNAscan be relatively-easily obtained by applying the recombinant DNAtechnology in general to insert the DNA into an appropriate vector.Examples of such a vector are those which have a property of replicatingin appropriate hosts. The following vectors can be arbitrarily used inthe present invention; pUC18, Bluescript® II SK(+), pKK223-3, and λgt·λCwhich require microorganisms of the genus Escherichia as hosts; pUB110,pTZ4, pC194, ρ11, φ1, and φ105 which require microorganisms of the genusBacillus as hosts; and pHY300PLK, pHV14, TRp7, YEp7, and pBS7 whichrequire at least two types of hosts. Referring to an example of methodfor inserting the present DNA into the vectors, an appropriate vectorand either the present DNA thus obtained or a DNA containing the presentDNA are cleaved with a restriction enzyme, and the formed DNA fragmentsand vector fragments are ligated. Examples of such a restriction enzymesuitably used are Acc I, Alu I, Bam HI, Bgl II, Bst XI, Eco RI, HindIII, Not I, Pst I, Sac I, Sal I, Sma I, Spe I, Xba I, and Xho I. In thecase of ligating the DNA and vector fragments, for example, chemicallysynthesized DNAS, having appropriate recognition sequences for therestriction enzymes, can be used. To ligate DNAs with others they arecontacted with DNA ligases intra- and extra-cellularly after annealing.

The present DNA includes those in the form of a transformant into whichthe DNA is introduced. Such a transformant is extremely usable to obtainthe present trehalose phosphorylase and DNA. For example, microorganismsof the genera Escherichia and Bacillus, actinomyces, and yeasts can bearbitrarily used as host microorganisms for the transformant. Thetransformant can be usually obtained by introducing the aforesaidrecombinant DNA into an appropriate host; when used a microorganism ofthe genus Escherichia, the microorganism and the recombinant DNA arecultured in the presence of calcium ion, while the competent cell andprotoplast methods are applied when used a microorganism of the genusBacillus. The aforesaid methods for preparing the present DNA are inthemselves conventional ones used in the art as described, for example,by J. Sumbruck et al. in "Molecular Cloning A Laboratory Manual", 2ndedition, published by Cold Spring Harbor Laboratory Press (1989).

The present process for producing trehalose phosphorylase ischaracterized in that it comprises culturing microorganisms whichproduce the present enzyme, and collecting the produced enzyme from thecultures. The genus and species of such microorganisms and cultivationmethods used in the present invention are not specifically restricted.Examples of such microorganisms include those which belong to the genusThermoanaerobium, preferably, Thermoanaerobium brockii, ATCC 35047, andtransformants obtained by introducing the present DNA into appropriatehost microorganisms.

Any natural- and synthetic-nutrient culture media can be used forculturing the microorganisms used in the present invention as long asthe microorganisms can grow therein and produce the present enzyme. Thecarbon sources used in the present invention are those which can beutilized by the microorganisms; for example, saccharides such asmaltose, trehalose, dextrins, and starches, and natural substances whichcontain saccharides such as molasses and yeast extracts can be used. Theconcentration of these carbon sources contained in the culture media ischosen depending on their types. For example, preferable saccharideconcentrations are not higher than 20 w/v %, and not higher than 5 w/v %with respect to the microorganisms' growth and proliferation. Thenitrogen sources used in the present invention are, for example,inorganic nitrogen-containing compounds such as ammonium salts andnitrates, and organic nitrogen-containing compounds such as urea, cornsteep liquor, casein, peptone, yeast extract, and meet extract. Ifnecessary, inorganic compounds, for example, salts of calcium,magnesium, potassium, sodium, phosphoric acid, manganese, zinc, iron,copper, molybdenum, and cobalt can be used in the present invention.

The microorganisms are anaerobically cultured under the conditionsselected from at temperatures of 50-80° C., preferably, 60-70° C., andat pHs of 5-8, preferably, 6.5-7.5. Any cultivation time can be used inthe present invention as long as it is sufficient for the growth of themicroorganisms, preferably, 10-50 hours. For the culture of the abovetransformants, they are generally cultured at temperatures of 20-65° C.and pHs of 2-9 for about 1-6 days under aerobic conditions byaeration-agitation method.

After culturing the microorganisms, the present enzyme can be collectedfrom the cultures. Because the enzyme activity may be generally presentintracellularly, intact and processed cells can be obtained as crudeenzymes. Whole cultures can be also used as crude enzymes. Conventionalsolid-liquid seperation methods can be used to separate cells andnutrient culture media; for example, methods to directly centrifuge thecultures, those to filtrate the cultures after adding filer aids to thecultures or after pre-coating, and those to filter the cultures usingmembranes such as plain filters and hollow fibers can be used. Theintact and processed cells per se can be used as crude enzymes, and ifnecessary, they can be prepared into partially purified enzymes.

The types of the processed cells include protein fractions of cells,immobilized preparations of intact and processed cells, and cells whichwere dried, lyophilized, and treated with surfactants, enzymes,ultrasonication, mechanical grinding, and mechanical pressure. Thepresent enzyme can be used in a crude or purified form, and theprocessed cells can be usually further treated with conventional methodsused for purifying enzymes, for example, salting out using ammoniumsulfate, sedimentation using acetone and alcohol, and membraneconcentration/dialysis using plain membranes and hollow fibers.

The intact and processed cells can be immobilized by conventionalmethods; for example, binding methods with ion exchangers, covalentbonding/adsorption methods with resins and membranes, and inclusionmethods using high molecular substances.

The crude enzymes can be used intact or may be purified by conventionalpurification methods. For example, the processed cells are salted outusing ammonium sulfate into crude enzymes, followed by dialyzing theenzymes and treating them successively with anion exchange columnchromatography using "DEAE-TOYOPEARL®", a cation exchangercommercialized by Tosoh Corporation, Tokyo, Japan, anion exchange columnchromatography using "CM-TOYOPEARL®" a resin commercialized by TosohCorporation, a hydrophobic column chromatography using"BUTYL-TOYOPEARL®", a hydrophobic resin commercialized by TosohCorporation, Tokyo, Japan, and gel filtration column chromatographyusing "ULTROGEL® AcA44 RESIN", a gel for gel filtration columnchromatography commercialized by Sepracor/IBF s.a. Villeneuve laGarenne, France, to obtain an electrophoretically single protein band ofenzyme.

The present trehalose phosphorylase activity is assayed as follows: Add0.2 ml of an enzyme solution to 2 ml of 20 mM phosphate buffer (pH 7.0)containing 1.0 w/v % trehalose as a substrate, incubate the solution at60° C. for 30 min, sample the reaction mixture in an amount of 0.5 ml,and incubate the sample at 100° C. for 10 min to suspend the enzymaticreaction. Add 0.5 ml of D-glucose oxidase/peroxidase reagent to theheated sample, stir the mixture, keep the mixture at 40° C. for 30 min,add 2.5 ml of 5-N hydrochloric acid, stir the resulting mixture, andmeasure the absorbance of the mixture at a wavelength of 525 nm. Oneunit of the enzyme activity is defined as the enzyme that forms onepmole of D-glucose per one minute. The activity of maltose- andkojibiose-phosphorylases can be assayed similarly as the same assay asindicated above except that the trehalose as a substrate is respectivelyreplaced with maltose and kojibiose.

In the present enzymatic reaction using the trehalose phosphorylase toproduce saccharide compositions containing glucosyl-transferredsaccharides, the enzyme is generally allowed to contact withβ-D-glucose-1-phosphoric acid as a saccharide donor along with otherappropriate saccharides as acceptors, for example, monosaccharides suchas D-xylose, D-galactose, G-glucose, D-fucose, and L-fucose to transformglucosyl group to the above reducing saccharides, resulting in aformation of glucosyl-D-xyloside, glucosyl-D-galactoside, trehalose,glucosyl-D-fucoside, and glucosyl-L-fucoside, respectively.

Commercially available β-D-glucose-1-phosphoric acid as a reagent can beused intact as a saccharide donor in the present invention, and it canbe prepared by contacting an appropriate phosphorylase with a saccharideas a substrate in the presence of an inorganic phosphoric acid and/orits salt; for example, it can be prepared by, in the presence of aninorganic phosphoric acid and/or its salt, contacting trehalosephosphorylase with trehalose, contacting maltose with maltosephosphorylase, or contacting kojibiose with kojibiose phosphorylase.When either of the above reactions of the phosphorylases that formβ-D-glucose-1-phosphoric acid is conducted in the same reaction systemusing the present trehalose phosphorylase to form glucosyl-transferredsaccharides, it can directly supply β-D-glucose-1-phosphoric acid to thesystem, resulting in a reduction of the production costs and asimplification of the production steps as advantageous features. Theinorganic phosphoric acid as mentioned above includes orthophosphoricacid and condensed phosphoric acid, and usually, the former acid ispreferably used. The salt of inorganic phosphoric acid includescompounds of phosphoric ion in general, derived from the above inorganicphosphoric acid, and usually, highly-water soluble sodium- andpotassium-salts of phosphoric acid are preferably used.

For example, the present enzyme can be used as the above trehalosephosphorylase, and commercially available bacterial maltosephosphorylases can be used as such. For the kojibiose phosphorylase, itcan be used the enzyme disclosed in Japanese Patent ApplicationNo.311,235/96, applied by the same applicant of the present invention,entitled "Kojibiose phosphorylase, its preparation and uses". Detaileddescriptions were in the specification; for example, a seed culture ofThermoanaeroblum brockii, ATCC 35047, was inoculated into the nutrientculture medium for the microorganism as described in "ATCC Catalogue ofBACTERIA AND BACTERIOPHAGES", 18th edition, pp.452-456 (1992), andcultured in the medium at 65° C. under anaerobic conditions, followed bycentrifuging the culture, disrupting the cells with ultrasonics, andcollecting the resulting supernatant to obtain the desired fraction withkojibiose phosphorylase activity.

The substrate concentration used in the glucosyl-transferred saccharideformation reaction using the present trehalose phosphorylase is notspecifically restricted. Generally, preferably used are solutionscontaining 1-20 w/w % (the wording "w/w %" will be abbreviated as "%"throughout the specification, unless specified otherwise) ofβ-D-glucose-1-phosphoric acid as a saccharide donor and 1-20% of anacceptor. As described above, when the β-D-glucose-1-phosphoric acidformation reaction by the action of an appropriate phosphorylase isconducted in the same reaction system of the above glucosyl-transferredsaccharide-forming reaction, the followings are recommendable: Whencontacting trehalose phosphorylase with its substrates, about 1-20%trehalose solutions are used in place of β-D-glucose-1-phosphoric acidas a substrate of the glucosyl-transferring reaction in the presence ofabout 0.5-20 mM of phosphates such as sodium dihydrogenphosphate. Whencontacting other phosphorylases with their substrates, about 1-20%maltose or kojibiose can be coexisted along with about 0.5-20 mM ofphosphates such as sodium dihydrogenphosphate in place of theβ-D-glucose-1-phosphoric acid as a substrate for theglucosyl-transferring reaction. Depending on the type of saccharidesused, either maltose- or kojibiose-phosphorylase can be preferablycoexisted in an amount of about 0.1-50 units/g saccharide, on a drysolid basis (d.s.b.).

The above reaction can be carried out at temperatures that do notinactivate the enzymes used in the presence of the substrates, i.e. upto about 70° C., preferably, about 15-65° C. The reaction pH can beusually adjusted to pHs of about 4.0-9.0, preferably, about 5.0-7.5. Thereaction time can be appropriately chosen depending on the enzymaticreaction rates, usually, it is about 0.1-100 hours when the enzymes areusually used in an amount of about 0.1-50 units/g substrate, d.s.b. Asdescribed above, when the β-D-glucose-1-phosphoric acid formationreaction by the action of different phosphorylases is conducted in thesame reaction system of the glucosyl-transferred saccharide-formingreaction, the reaction temperatures and pHs are preferably set to thosewhich do not inactivate the phosphorylases used depending on theirthermal stabilities.

Thus, the resulting reaction mixtures contain glucosyl-transferredsaccharides which correspond to the saccharides used as substrates. Theyields of glucosyl-transferred saccharides are varied depending on thesubstrate concentrations, types of substrates, and reaction conditionsused in the enzymatic reactions. For example, in the case of using 10%trehalose and 5% D-galactose are used as substrates in the presence ofan inorganic phosphoric acid, glucosylgalactoside is formed in a yieldof about 30%. Throughout the specification, the yields ofglucosyl-transferred saccharides mean their percentages (%) of theformed saccharides to the total saccharides in reaction mixtures, d.s.b.

To increase the content of glucosyl-transferred saccharides in thereaction mixtures to the highest possible level, enzyme sources, whichdecompose and remove D-glucose formed in the reaction mixtures, can beadvantageously coexisted in the mixtures to promote thesaccharide-transferring reactions. Such a technique can besatisfactorily used to decompose and remove D-glucose as a by-productformed during the reaction processes and to promote thesaccharide-transferring reaction when the above β-D-glucose-1-phosphoricacid formation reaction using an appropriate phosphorylase is carriedout in the same reaction system of the glucosyl-transferring reaction todirectly supply saccharide donors.

The enzyme sources are microorganisms which have a D-glucose decomposingactivity, cultures of such microorganisms, their intact and processedcells, and enzymes with a D-glucose decomposing activity. Anymicroorganisms can be used as long as they have a relatively-highD-glucose decomposing activity but have no or substantially no activityof decomposing the formed transferred saccharides; preferable ones areyeasts. Any enzymes such as glucose oxidase, catalase, pyranose oxidase,glucose dehydrogenase, glucokinase, and hexokinase can be used. Amongthese enzymes, the glucose oxidase and catalase can be preferably used.

The reaction mixtures containing the glucosyl-transferred saccharidesthus produced can be in a conventional manner filtered and centrifugedto remove impurities, then subjected to purification steps such asdecoloration with activated charcoals, and desalting with ion exchangersin H- and OH-form, and concentrated into syrupy products. If necessary,the syrupy products can be arbitrarily dried by spray drying, etc., intopowdery products.

The present saccharide compositions containing glucosyl-transferredsaccharides can be processed into products rich in the saccharides byseparating the saccharides from reaction mixtures and purifying theresulting mixtures. Examples of such separation methods are those whichseparate and remove impurities; fermentation methods using yeasts toremove monosaccharides or yeast fermentation methods, and methods toremove concomitant saccharides such as membrane filtrations, columnchromatographies, and methods comprising adjusting the reaction mixturesto alkaline pHs, and heating the mixtures to decompose reducingsaccharides. More particularly, advantageously used on an industrialscale are methods for removing concomitant saccharides to collectfractions rich in the desired glucosyl-transferred saccharides byremoving concomitant saccharides on column chromatographies usingstrong-acid cation exchange resins as disclosed in Japanese Patent KokaiNos.23,799/83 and 72,598/83. In this case, conventional fixed-bed,moving-bed, and semi-moving methods can be arbitrarily used.

The solutions separated from impurities can be in a conventional mannerfiltered and centrifuged to remove insoluble substances, subjected topurification steps such as decoloration with activated charcoals, anddesalting with ion exchange resins in H- and OH-form, and concentratedinto syrupy products. If necessary, the syrupy products can be dried bymethods such as spray drying into powdery products.

The present saccharide compositions containing glucosyl-transferredsaccharides thus obtained usually contain at least 5%, preferably, atleast 10% of the glucosyl-transferred saccharides, d.s.b.

The present saccharide compositions containing glucosyl-transferredsaccharides have a satisfactory taste and sweetness, osmosis-controllingability, humectancy, gloss-imparting ability, crystallization-preventingability, and retrogradation-preventing ability, anti-cariosity,growth-promoting activity for bifid bacteria, andmineral-absorption-promoting activity. Because of these satisfactoryproperties and functions, the present saccharide compositions can bearbitrarily used widely in compositions such as food products, tobaccos,cigarettes, feeds, pet foods, cosmetics, pharmaceuticals, shaped bodies,daily foods and products, products of forestry and fisheries, reagents,and products for chemical industries.

The present saccharide compositions containing glucosyl-transferredsaccharides can be used intact as a seasoning for sweetening. Ifnecessary, the present saccharide compositions can be used together withadequate amounts of one or more other sweeteners, for example, powderedsyrup, glucose, maltose, trehalose, sucrose, isomerized sugar, honey,maple sugar, sorbitol, maltitol, lactitol, dihydrocharcone, stevioside,α-glycosyl stevioside, rebaudioside, glycyrrhizin,L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, andalanine, as well as fillers such as dextrins, starches, and lactose.

The present saccharide compositions have a sweetness which wellharmonizes with substances having sourness, acidity, saltiness,bitterness, astringency, and deliciousness, as well as a satisfactoryacid- and heat-tolerance. Thus, they can be arbitrarily used in foodproducts in general as a sweetener, taste-improving agent, andquality-improving agent.

The present saccharide compositions can be used in seasonings such assoy sauces, powdered soy sauces, "miso", "funmatsu-miso" (a powderedmiso), "moromi" (a refined sake), "hishio" (a refined soy sauce),"furikake" (a seasoned fish meal), mayonnaise, dressings, vinegars,"sanbai-zu" (a sauce of sugar, soy sauce and vinegar),"funmatsu-sushi-su" (powdered vinegar for sushi), "chuka-no-moto" (aninstant mix for Chinese dish), "tentsuyu" (a sauce for Japanese deep-fatfried food), "mentsuyu" (a sauce for Japanese vermicelli), sauces,catsups, premixes for pickles and pickled products such as"takuan-zukeno-moto" (a premix for pickled radish), and"hakusai-zuke-nomoto" (a premix for fresh white rape pickles),"yakiniku-notare" (a sauce for Japanese grilled meat), curry roux,instant stew mixes, instant soup mixes, "dashi-no-moto" (an instantstock mix), mixed seasonings, "mirin" (a sweet sake), "shin-mirin" (asynthetic mirin), table syrups, and coffee syrups.

The present saccharide composition can be freely used for sweetening"wagashi" (Japanese cakes) such as "senbei" (a rice cracker),"arare-mochi" (a rice-cake cube), "okoshi" (a millet-and-rice cake),"mochi" (a rice paste), "manju" (a bun with a bean-jam), "uiro" (a sweetrice jelly), "an" (a bean jam), "yokan" (a sweet jelly of beans),"mizu-yokan" (a soft adzuki-bean jelly), "kingyoku" (a kind of yokan),jellies, pao de Castellas and "amedama" (a Japanese toffee);confectioneries such as buns, biscuits, crackers, cookie, pies,puddings, butter creams, custard creams, cream puffs, waffles, spongecakes, doughnuts, chocolates, chewing gums, caramels and candies; frozendesserts such as ice creams and sherbets; syrups such as"kajitsu-no-syrup-zuke" (a preserved fruit), and "korimitsu" (a sugarsyrup for shaved ice); pastes such as flour pastes, peanut pastes, fruitpastes, and spreads; processed fruits and vegetables such as jams,marmalades, "syrup-zuke" (fruit pickles), and "toka" (conserves);pickles and pickled products such as "fukujin-zuke" (red colored radishpickles), "bettara-zuke" (a kind of whole fresh radish pickles),"senmai-zuke" (a kind of sliced fresh radish pickles), and "rakkyo-zuke"(pickled shallots); meat products such as hams and sausages; products offish meats such as fish hams, fish sausages, "kamaboko" (a steamed fishpaste), "chikuwa" (a kind of fish paste), and "tenpura" (a Japanesedeep-fat fried fish paste); "chinmi" (a relish) such as"uni-no-shiokara" (salted guts of sea urchin), "ika-no-shiokara" (saltedguts of squid), "su-konbu" (processed tangle), "saki-surume" (driedsquid strips), and "fugu-no-mirin-boshi" (a dried mirin-seasonedswellfish); "tsukudani" (a food boiled down in soy sauce) such as thoseof layers, edible wild plants, dried squids, fishes, and shellfishes;daily dishes such as "nimame" (cooked beans), potato salads, and"konbu-maki" (a tangle roll); milk products; canned and bottled productssuch as those of meats, fish meats, fruits, and vegetables; alcoholicbeverages such as sakes, synthetic sakes, wines and liquors; soft drinkssuch as coffees, teas, cocoas, juices, carbonated beverages, sour milkbeverages, and beverages containing lactic acid bacteria; instant foodproducts such as instant pudding mixes, instant hot cake mixes, and"sokuseki-shiruko " (an instant mix of adzuki-bean soup with rice cake),and instant soup mixes; and foods such as baby foods, foods for therapy,beverages supplemented with nutritions, cooked rice products, noodles,and frozen foods; as well as for improving the tastes and qualities ofthe above food products.

The present saccharide compositions can be also used in feeds and petfoods for animals such as domestic animals, poultry, and fishes toimprove their taste preferences. The present saccharide compositions canbe arbitrarily used as a sweetener, taste-improving agent, andquality-improving agent in other compositions in a paste and liquid formsuch as tobaccos, cigarettes, dentifrices, lipsticks, rouges, lipcreams, internal medicines, tablets, troches, cod liver oils in the formof drops, cachous, oral refrigerants, gargles, cosmetics, andpharmaceuticals.

The present saccharide compositions can be used as a quality-improvingagent and stabilizer in biologically active substances susceptible tolose their effective ingredients and activities, as well as in healthfoods and pharmaceuticals containing the above biologically activesubstances. Examples of such biologically active substances aresolutions of cytokines such as α-, β- and γ-interferons, tumor necrosisfactor-α (TNF-α), tumor necrosis factor-β (TNF-β), macrophage migrationinhibitory factor, colony-stimulating factor, transfer factor, andinterleukins 1, 2, 6, 12, 15 and 18; hormones such as insulin, growthhormone, prolactin, erythropoietin, tissue plasminogen activator,follicle-stimulating hormone, and placental hormone; biologicalpreparations such as BCG vaccine, Japanese encephalitis vaccine, measlesvaccine, live polio vaccine, smallpox vaccine, tetanus toxoid,Trimeresurus antitoxin, and human immunoglobulin; antibiotics such aspenicillin, erythromycin, chloramphenicol, tetracycline, streptomycin,and kanamycin sulfate; vitamins such as thiamine, riboflavin, L-ascorbicacid, cod liver oil, carotenoid, ergosterol, and tocopherol; enzymessuch as lipase, elastase, urokinase, protease, β-amylase, isoamylase,glucanase, and lactase; extracts such as ginseng extract, snappingturtle extract, chlorella extract, aloe extract, and propolis extract;viable microorganisms such as viruses, lactic acid bacteria, and yeasts;and other biologically active substances such as royal jelly. By usingthe present saccharide compositions, the aforementioned biologicallyactive substances are arbitrary prepared into health foods andpharmaceuticals with a satisfactorily-high stability and quality withoutfear of losing or inactivating their effective ingredients andactivities.

As described above, the wording "compositions" as referred to in thepresent invention include orally- and parenterally-usable food products,cosmetics, and pharmaceuticals, as well as daily products, products offorestry and fisheries, reagents, and products for chemical industries.

Methods to incorporate the present saccharide compositions into theabove compositions include conventional methods, for example, mixing,kneading, dissolving, melting, soaking, permeating, sprinkling,applying, coating, spraying, injecting, and solidifying. The presentsaccharide composition is usually incorporated into the compositions inan amount of 0.1% or more, preferably, 0.5% or more.

The following experiments explain the present invention in more detail:

EXPERIMENT 1

Preparation of trehalose phosphorylase

According to the preparation of the medium for Thermoanaeroblum brockiias disclosed in "ATCC Catalogue of BACTERIA AND BACTERIOPHAGES", 18thedition, pp.452-456 (1992), except for replacing 0.5 w/v % glucose with0.5 w/v % trehalose as a carbon source, a medium was prepared, and 100ml aliquots of the medium were placed in 100-ml pressure bottles,followed by inoculating a seed of Thermoanaeroblum brockii, ATCC 35047,and allowing to stand at 60° C. for 48 hours for a seed culture.

About 10 L aliquots of a fresh preparation of the same nutrient culturemedium as used for preparing the seed culture were placed in four 11-lstainless steel bottles, sterilized by heating, cooled to 60° C., andinoculated with one v/v % of the seed culture to the culture medium,followed by the stationary culture at 60° C. for about 40 hours.

About 40 L of the resultant pooled cultures were centrifuged to obtain92 g wet cells which were then suspended in 10 mM phosphate buffer,ultrasonicated, and centrifuged to obtain a supernatant of the disruptedcell suspension. The supernatant had an activity of 0.3 unit/ml oftrehalose phosphorylase.

EXPERIMENT 2

Purification of trehalose phosphorylase

The supernatant in Experiment 1 was concentrated using a UF membraneinto an about 360 ml of enzyme concentrate having an activity of about30 units/ml of trehalose phosphorylase.

Three hundred ml of the enzyme concentrate was dialyzed against 10 mMphosphate buffer (pH 7.0) for 24 hours, and centrifuged to removeinsoluble substances. Three hundred and eighty ml of the resultingsupernatant was subjected to ion exchange column chromatography using380 ml of "DEAE-TOYOPEARL® 650 GEL", a gel for ion exchange columnchromatography commercialized by Tosoh Corporation, Tokyo, Japan.

The present trehalose phosphorylase was allowed to be adsorbed on thegel, and eluted from the column by feeding a linear gradient of sodiumchloride increasing from 0M to 0.5M. Fractions with the enzyme activity,eluted at about 0.1M sodium chloride, were collected and pooled, and theenzyme in the pooled solution was then purified as follows: Dialyze thesolution against a fresh preparation of the same buffer containing 1.5Mammonium sulfate, centrifuge the dialyzed solution to remove insolublesubstances, and subject the supernatant to hydrophobic columnchromatography using 100 ml of "BUTYL-TOYOPEARL® 650 GEL". Elute thetrehalose phosphorylase adsorbed on the gel with a linear gradient ofammonium sulfate decreasing from 1.5M to 0.5M, and collect fractionswith the enzyme activity.

The fractions were pooled and subjected to gel filtration chromatographyusing 300 ml of "ULTROGEL® AcA44 RESIN", a gel for gel filtration columnchromatography commercialized by Sepracor/IBF s.a. Villeneuve laGarenne, France, followed by collecting fractions with the enzymeactivity.

The yield of the purified enzyme specimen, obtained by the abovepurification steps, was about 25% with respect to the enzyme activity ofthe supernatant of the disrupted cell suspension. The enzyme specimenhad a specific activity of 78.2 units/mg protein. Protein was quantifiedaccording to the Lowry method using calf serum albumin as a standardprotein.

Examination for the purity of the specimen on gel electrophoresis using7.5 w/v % polyacrylamide revealed that the specimen was arelatively-high purity protein detected as a single protein band.

EXPERIMENT 3

Property of trehalose phosphorylase

The trehalose phosphorylase specimen in Experiment 2 was subjected toSDS-PAGE using 10 w/v % gel. Comparing with marker proteins,commercialized by Japan Bio-Rad Laboratories, Tokyo, Japan, which wereelectrophoresed in parallel, the molecular weight of the enzyme wasmeasured, revealing that it had a molecular weight of 88,000±5,000daltons and gave a molecular weight of 190,000±10,000 daltons on gelfiltration using a column, 7.5 mm in diameter and 600 mm in length,packed with "TSKgel G4000SW", a gel for gel filtration commercialized byTosoh Corporation, Tokyo, Japan.

The purified trehalose phosphorylase was subjected to polyacrylamide gelelectrophoresis using 2 w/v e "AMPHOLINE", an ampholyte commercializedby Pharmacia LKB Biotechnology AB, Uppsala, Sweden, followed bymeasuring the pHs of protein bands and gels, revealing that the enzymehad a pI of 5.4±0.5.

Influences of temperatures and pHs on the present trehalosephosphorylase activity were studied in accordance with the assay forenzyme activity. To study the influence of temperatures, the enzyme wasreacted at temperatures of about 50-85° C. in place of 60° C. as used inthe enzyme assay. In the case of studying the influence of pHs, theenzyme was reacted at pHs of about 4-9 in place of the buffer's pH asused in the assay for the enzyme activity. In both cases, the enzymaticreactions were suspended similarly as in the enzyme assay, followed byquantifying the formed glucose. These results were in FIGS. 1 and 2which were respectively the data for influences of temperatures and pHs,and expressed by relative values to the maxima. The enzyme had anoptimum temperature of about 70° C. when incubated at pH 7.0 for 30 min,and the optimum pH was about 7.0-7.5 when incubated at 60° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe enzyme dissolved in 10 mM phosphate buffer (pH 7.0) at a temperatureof about 40-85° C. for one hour, cooling the incubated enzyme, andassaying for the residual enzyme activity according to the enzyme assay.The pH stability of the enzyme was determined by dissolving the enzymein buffers with different pHs of about 4-10, keeping each enzymesolution at 4° C. for 24 hours, adjusting each solution to give a pH of7.0, and assaying for the residual enzyme activity according to theenzyme assay. These results were in FIGS. 3 and 4 which wererespectively the data for thermal and pH stabilities of the enzyme andexpressed by relative values to the maxima. The enzyme had a thermalstability of up to about 60° C. and a pH stability of about 6.0-9.0. Theenzyme activity was inhibited by one mM Cu⁺⁺, Pb⁺⁺, Zn⁺⁺, Hg⁺⁺, Mg⁺⁺, orMn⁺⁺.

EXPERIMENT 4

Partial amino acid sequence of trehalose phosphorylase Experiment 4-(1)N-Terminal amino acid sequence

A portion of a purified enzyme specimen, obtained by the method inExperiment 2, was dialyzed against distilled water, and about 40 μg ofthe dialyzed enzyme by protein weight was used as a sample for analyzingthe N-terminal amino acid sequence. "PROTEIN SEQUENCER MODEL 473A", anapparatus commercialized by Applied Biosystems, Inc., Foster City, USA,was used to analyze up to five amino acid resides from the N-terminus.The analyzed partial amino acid sequence was SEQ ID NO:1. More preciseanalysis using a fresh preparation of the same enzyme specimen revealedthat the enzyme has the amino acid sequence of SEQ ID NO:6 at theN-terminus.

EXPERIMENT 4-(2)

Internal partial amino acid sequence

A portion of a purified enzyme specimen, obtained by the method inExperiment 2, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), andthe dialyzed solution was diluted with a fresh preparation of the samebuffer to give a protein concentration of one mg/ml. One ml of theresulting solution was admixed with 10 μg of lysyl endopeptidase, anenzyme specimen commercialized by Wako Pure Chemical Industries, Ltd.,Tokyo, Japan, and subjected to an enzymatic reaction at 30° C. for 22hours to form peptides. Reversed phase HPLC (high-performance liquidchromatography) was performed to isolate the peptides under theconditions of using "μBONDASPHERE C-18 COLUMN (2.1 mm in diameter and150 mm in length)", a column for reversed phase HPLC commercialized byWaters Chromatography, Div., MILLIPORE Corp., Milford, Mass., USA, flowrate of 0.9 ml/min, ambient temperature, and a linear gradient ofacetonitrile increasing from 0 v/v % to 48 v/v t in 0.1 v/v %trifluoroacetic acid over 120 min. Peptides eluted from the column weredetected by measuring their absorbances at 210 nm. Two peptides, i.e.,TP10 with a retention time of 66 min and TP14 with a retention time of86 min, which were clearly separated from others, were separatelycollected, dried in vacuo, and dissolved in 200 μl of a solution of 0.1v/v % trifluoroacetic acid and 50 v/v % acetonitrile. Each peptide wasanalyzed on a protein sequencer to reveal up to five amino acid residuesfrom the N-terminus. The TP10 and TP14 gave the amino acid sequences ofSEQ ID NO:2 and SEQ ID NO:3, respectively. More precise analysis of afresh preparation of the same enzyme specimen by the same analysisrevealed that the TP14 has SEQ ID NO:7 at the N-terminus.

EXPERIMENT 5

Substrate specificity for saccharide-hydrolyzing reaction by trehalosephosphorylase

An aqueous solution of a saccharide selected from D-glucose, maltose,sucrose, lactose, trehalose, neotrehalose, cellobiose, melibiose,kojibiose, isomaltose, sophorose, gentibiose, nigerose, laminaribiose,maltopentaose, and 4-O-α-D-glucosyltrehalose was mixed with 10 units/gsaccharide, d.s.b., of a purified trehalose phosphorylase obtained bythe method in Experiment 2, and incubated in the presence of 5 mMdisodium hydrogenphosphate at 60° C. and pH 7.0 for 24 hours. Thesaccharide concentration of each reaction solution was 2 w/v %.Pre-reaction solutions and post-reaction mixtures were subjected to thinlayer chromatography (hereinafter abbreviated as "TLC") using "KIESELGEL60 (20×20 cm)", an aluminum plate for TLC commercialized by Merck & Co.,Inc., Rahway, USA. Samples were developed once at ambient temperature onthe plate using 1-butanol/pyridine/water (=7:3:1 by volume) as adeveloping solvent system, and the plate was sprayed with 20 v/v %sulfuric acid/methanol solution, and heated at 110° C. for about 10 minfor coloration. Comparing spots of the solutions and mixtures detectedon the plates, it was checked whether the enzyme acted on thesaccharides. The results were in Table 1:

                  TABLE 1    ______________________________________    Substrate          Decomposition    ______________________________________    D-Glucose          -    Maltose            -    Sucrose            -    Lactose            -    Trehalose          +    Neotrehalose       -    Cellobiose         -    Melibiose          -    Kojibiose          -    Isomaltose         -    Sophorose          -    Gentibiose         -    Nigerose           -    Laminaribiose      -    Maltopentaose      -    4-O-α-D-glucosyltrehalose                       -    ______________________________________     Note)     +: Hydrolyzed by the action of the present trehalose phosphorylase.     -: Not hydrolyzed by the action of the present trehalose phosphorylase.

As shown in Table 1, it was found that the present trehalosephosphorylase showed a strong substrate specificity to trehalose andacted on it to form D-glucose and β-D-glucose1-phosphoric acid, but didnot act on other saccharides.

EXPERIMENT 6

Specificity to acceptor in glucosyl-transferring saccharide-formingreaction by trehalose phosphorylase

An aqueous solution, which dissolved in an equal amount, by dry weight,of β-D-glucose-1-phosphoric acid as a saccharide donor and one of themonosaccharides, oligosaccharides, and sugar alcohols as acceptors inTable 2, was admixed with 10 units/g β-D-glucose-1-phosphoric acid of apurified trehalose phosphorylase obtained by the method in Experiment 2,and enzymatically reacted at 60° C. and pH 7.0 for 24 hours. Similarlyas in Experiment 5, the pre-reaction solutions and the post-reactionmixtures were subjected to TLC, followed by coloring the plates.Comparing spots of the samples of the solutions and mixtures detected onthe plates, it was judged whether transferred saccharides were formedbased on newly detected spots of the post reaction mixtures. Bymacroscopically observing the coloration degree of the newly detectedspots, the yield of the transferred saccharides was relativelyevaluated. The results were in Table 2:

                  TABLE 2    ______________________________________    Acceptor              Formation of glucosyl-    Classification              Name            transferred saccharide    ______________________________________    Aldopentose              D-Xylose        +++              L-Xylose        -              D-Ribose        -    Aldohexose              D-Galactose     ++              D-Glucose       +++              D-Mannose       +    Ketohexose              L-Sorbose       -              D-Fructose      -    Deoxysugar              2-Deoxyglucose  ++              D-Fucose        +++              L-Fucose        +++    Glucoside α-Methyl glucoside                              -              β-Methyl glucoside                              -    Sugar alcohol              Sorbitol        -    Amino sugar              Glucosamine     ++              N-Acetyl glucosamine                              ++    Disaccharide              Maltose         -              Lactose         -              Isomaltose      -              Cellobiose      -              Sucrose         -    ______________________________________     Note)     -: No glucosyltransferred saccharide formed.     +: Glucosyltransferred saccharide was formed in a relativelysmall amount.     ++: Glucosyltransferred saccharide was formed in a relativelylarge amount     +++: Glucosyltransferred saccharide was formed in a considerablylarge     amount.

As shown in Table 2, it was revealed that the present trehalosephosphorylase forms glucosyl-transferred saccharides by effectivelytransferring glucosyl group from β-D-glucose-1-phosphoric acid as asaccharide donor to reducing aldoses as acceptors such asmonosaccharides such as D-xylose, D-galactose, D-glucose,2-deoxy-D-glucose, D-fucose, L-fucose, glucosamine, and N-acetylglucosamine. Considering the substrate specificity of the presentenzyme, these glucosyl-transferred saccharides were judged to benon-reducing saccharides. No glucosyl-transferred saccharide wasobtained when α-D-glucose-1-phosphoric acid was used as a saccharidedonor in place of β-D-glucose-1-phosphoric acid.

Some of the glucosyl-transferred saccharides, which were revealed inExperiment 6 to be formed via the saccharide-transferring reaction bythe present trehalose phosphorylase, will be explained on their detailedstructures with reference to the following Experiments 7 and 8.

EXPERIMENT 7

Glucosyl-transferred saccharide from D-glucose andβ-D-glucose-1-phosphoric acid

A portion of the reaction mixture, obtained by using as substratesD-glucose and β-D-glucose-1-phosphoric acid in Experiment 6, was dilutedwith 10 mM phosphate buffer (pH 7.0) to give a concentration of onepercent, admixed with 0.5 ml of a trehalase specimen commercialized bySigma Chemical Company, St. Louis, USA, and enzymatically reacted at 45°C. for 20 hours. Each portion of the reaction mixtures treated oruntreated with trehalase was dried, dissolved in pyridine, andtrimethyl-silylated. The resulting products were analyzed on gaschromatography (hereinafter abbreviated as "GLC") under the conditionsof using a stainless steel column, 3 mm in diameter and 2 m in length,packed with 2% "SILICONE OV-17/CHROMOSOLB W", a resin for GLCcommercialized by GL Sciences Inc., Tokyo, Japan, nitrogen gas as acarrier gas, flow rate of 40 ml/min, column oven temperatures of160-320° C., and heating-up rate of 7.5° C./min. Saccharide componentswere detected by a hydrogen flame ionization detector.

As a result, it was revealed that the retention time of a peak for aglucosyl-transferred saccharide, formed from D-glucose andβ-D-glucose-1-phosphoric acid by the action of the present trehalosephosphorylase, was agreed with that of authentic trehalose, and that thetrehalase treatment diminished the peak and formed D-glucose.Considering the substrate specificity of trehalase, it was speculatedthat the glucosyl-transferred saccharide was trehalose.

EXPERIMENT 8

Glucosyl-D-galactoside

To identify the glucosyl-transferred saccharide formed via thesaccharide-transferring reaction to D-galactose by the present trehalosephosphorylase, the glucosyl-transferred saccharide was prepared,isolated, and examined for structure. The procedures were as follows:Provide an aqueous solution containing 5% trehalose, 2.5% D-galactose,and 5 mM sodium dihydrogenphosphate, adjust the aqueous solution to givea pH of 5.0, add to the solution 15 units/g β-D-glucose-1-phosphoricacid of a purified trehalose phosphorylase obtained by the method inExperiment 2, enzymatically react the solution at 60° C. for 72 hours,heat the reaction mixture at 100° C. for 10 min to inactivate theremaining enzyme, and analyze on GLC a sample from the resulting mixtureaccording to the method in Experiment 7. As a result, it was confirmedthat the reaction mixture contained a relatively-large amount of asubstance with a retention time differing from those of trehalose,D-galactose, D-glucose, and β-D-glucose-1-phosphoric acid, estimatingthat it was the glucosyl-transferred saccharide. Based on the data fromGLC, the yield of the saccharide was about 30%. The remaining reactionmixture was adjusted to give a pH of 7.0, admixed with 25 units/gtrehalose remained, and enzymatically reacted at 45° C. for 20 hours todecompose trehalose remaining in the reaction mixture. The resultant washeated at 100° C. for 10 min to inactivate the remaining trehalase,decolored with activated charcoals, filtered, desalted and purifiedusing ion exchange resins in H- and OH-form, concentrated up to give aconcentration of about 50%, and subjected to the column chromatographybelow, followed by collecting fractions rich in the glucosyl-transferredsaccharide.

The resin used for fractionation was "XT-1016", an alkali metalstrong-acid cation exchange resin, Na-form, polymerization degree of 4%,commercialized by Tokyo Organic Chemical Industries, Ltd., Tokyo, Japan,and the resin was suspended in water, packed in four jacketed-stainlesssteel columns, 3 cm in diameter and one meter in length each, which werecascaded in series to give a total gel-bed depth of about 4 m. Keepingthe inner column temperature at 40° C., a saccharide solution was fed tothe columns in a volume of 5 v/v % to the resin, followed by feeding tothe columns water heated to 40° C. at a flow rate of SV (space velocity)0.15 to fractionate the saccharide solution and collecting fractionsrich in the glucosyl-transferred saccharide.

The fractions were pooled, desalted, purified, and concentrated into anabout 40% concentrate which was then chromatographed on a column packedwith "YMC-Pack OSD", an octadecyl silica gel commercialized by YMC Co.,Ltd., Kyoto, Japan, to collect fractions containing theglucosyl-transferred saccharide. The fractions were pooled andconcentrated into an about 40% concentrate which was then re-applied tothe above column chromatography. The resulting solution rich in theglucosyl-transferred saccharide was desalted, purified, concentrated,and dried in vacuo to obtain a powdery product containing the saccharidein a yield of about 20%, d.s.b., to the material saccharide used in theenzymatic reaction. In accordance with the method in Experiment 7, thepowdery product was analyzed on GLC, revealing that it contained about98% of the glucosyl-transferred saccharide, d.s.b.

The powdery product rich in the glucosyl-transferred saccharide wassubjected to GLC analysis after decomposed with acids, revealing thatthe saccharide produced D-glucose and D-galactose in a molar ratio ofabout 1:1 when decomposed with acids. The powdery product wasmethylated, hydrolyzed by acids, reduced, and acetylated to obtainpartial methylhexytolacetate which was then analyzed on GLC to detect2,3,4,6-tetra-O-methyl-1,5-di-O-acetylglucitol, and2,3,4,6-tetra-O-methyl-1,5-di-O-acetylgalactitol. The data indicatesthat the glucosyl-transferred saccharide is composed of D-glucose andD-galactose in a molar ratio of 1:1 where both the OH-group at C-1 ofD-glucose and the OH-group at C-1 of D-galactose relate to the bondingbetween these saccharides.

To examine the structure of the glucosylgalactoside in more detail, thesaccharide was measured for specific rotation and ¹³ C-NMR spectrum. Asa result, it gave α!^(D) ₂₀ =+223° (c=0.97, H₂ O) and ¹³ C-NMR spectra(100 MHz, D₂ O):σppm from TSP of 96.16, 95.97, 75.37, 74.94, 74.14,73.90, 72.53, 72.11, 71.80, 70.76, 64.03, and 63.37. These data werenearly agreed with the authentic data of a chemically synthesizedcompound, α-D-galactopyranosyl α-D-glucopyranoside, and this confirmedthat the glucosylgalactoside was glucosyl-D-galactoside, i.e.,α-D-galactopyranosyl α-D-glucopyranoside, a disaccharide composed ofD-glucose and D-galactose bound in an α-1,1 linkage.

EXPERIMENT 9

Acute toxicity test

Acute toxicity tests of a powdery product rich in glucosyl-D-galactosideobtained by the method in Experiment 8, a powdery product rich inglucosyl-D-xyloside obtained by the method in Example A-12, a powderyproduct rich in glucosyl-D-fucoside obtained by the method in ExampleA-14, and a powdery product rich in glucosyl-L-fucoside obtained by themethod in Example A-15 were respectively tested in 7-week-old dd-strainmice by administering orally. As a result, no mouse died even whenadministered with their maximum doses, i.e., 50 g/kg mouse by weight.The data indicates that these saccharides are extremely low in toxicity.

Example A explains the present trehalose phosphorylase, DNA encoding theenzyme, and process for producing saccharides containingglucosyl-transferred saccharides prepared by using the enzyme, and ofcourse, these embodiments do not limit the present invention:

EXAMPLE A-1

Enzyme solution

Thermoanaerobium brockii, ATCC 35047, was cultured for about 30 hours ina fresh preparation of the same medium as used in Experiment 1, exceptfor setting the temperature to 65° C., using a fermenter under anaerobicconditions according to the method in Experiment 1. The resultingculture was centrifuged to obtain cells which were then disrupted byultrasonic and centrifuged. The supernatant was fed to a column packedwith "DEAE-TOYOPEARL® GEL" to be adsorbed thereupon, and eluted from thecolumn by feeding an aqueous linear gradient of sodium chlorideincreasing from 0M to 0.5M, followed by collecting fractions with atrehalose phosphorylase activity eluted at about 0.1M sodium chloride.The fractions were pooled and concentrated with an ultrafiltrationmembrane to obtain an enzyme solution with about 20 units/ml oftrehalose phosphorylase in a yield of about 40% to the total activity ofthe material culture.

EXAMPLE A-2

Preparation of DNA

According to the method in Experiment 1, a seed of Thermoanaeroblumbrockii, ATCC 35047, was inoculated into 11 L of a fresh preparation ofthe same nutrient culture medium as used in Experiment 1, and culturedat 60° C. for 24 hours. The proliferated cells were separated from theculture by centrifugation, suspended in an adequate amount ofTris-EDTA-saline buffer (hereinafter abbreviated as "TES buffer") (pH8.0), admixed with 0.05 w/v % of lysozyme to the cell suspension, andincubated at 37° C. for 30 min. Thereafter, the enzyme-treated mixturewas freezed at -80° C. for one hour, and admixed successively with TESbuffer (pH 9.0) and a mixture solution of TES buffer-phenol heated to60° C., followed by sufficiently stirring and cooling the mixture,centrifuging the resultant, and collecting the formed upper-layer.Twofold volumes of cooled ethanol was added to the layer, and the formedsediment was collected, dissolved in an adequate amount of SSC buffer(pH 7.1), admixed with 7.5 μg ribonuclease and 125 μg protease, andincubated at 37° C. for one hour. To the resulting mixture was added amixture solution of chloroform and isoamyl alcohol, followed by stirringand allowing to stand the mixture, and collecting the formedupper-layer. After adding cooled ethanol to the layer, the formedsediment was collected, rinsed with 70 v/v % cooled ethanol, and driedin vacuo to obtain a DNA. The DNA was dissolved in SSC buffer (pH 7.1)to give a concentration of about one mg/ml, and freezed at -80° C.

EXAMPLE A-3

Preparation of transformant and recombinant DNA

One ml of the DNA solution in Example A-2 was placed in a container,admixed with about 20 units of a restriction enzyme, Alu I, andincubated at 37° C. for 30 min to partially digest the DNA. Theresulting mixture was subjected to sucrose density ultrafiltration tocollect a DNA fragment of about 2,000-5,000 base pairs. In parallel,"Bluescript® II SK(+)", a plasmid vector commercialized by StratageneCloning Systems, California, USA, was completely cleaved with arestriction enzyme, Sma I, and 0.3 μg of the cleaved vector and about 3μg of the DNA fragment were ligated using "DNA LIGATION KIT", TakaraShuzo Co., Ltd., Otsu, Shiga, Japan, according to the procedure attachedto the kit. Using the recombinant DNA thus obtained, 100 pg of"EPICURIAN COLI® XL1-BLUE", a microorganism of the species Escherichiacoli commercialized by Stratagene Cloning Systems, California, USA, wastransformed by conventional competent cell method to obtain a genelibrary.

The resulting transformants as a gene library were inoculated into agarplate (pH 7.0), prepared in a usual manner, containing 10 g/l trypton, 5g/l yeast extract, 5 g/l sodium chloride, 75 mg/l sodium salt ofampicillin, and 50 mg/l 5-bromo-4-chloro-3-indolyl-β-galactoside, andcultured at 37° C. for 18 hours, followed by fixing about 5,000 whitecolonies, formed on the plate, onto "HYBOND-N+", a nylon filmcommercialized by Amersham Corp., Div. Amersham International,Arlington, Heights, USA. Based on the amino acids 9-15 in the N-terminalregion of SEQ ID NO:6 revealed in Experiment 4, an oligonucleotide witha nucleotide sequence, represented by 5'-TAYCCNTTYGARGAYTGGGT-3'(SEQ IDNO:9, was chemically synthesized, and labelled with γ-³² P!ATP and T4polynucleotide kinase to obtain a synthesized DNA as a first probe.Among the colonies fixed on the nylon film, three colonies stronglyhybridized with the first probe was selected by applying conventionalcolony-hybridization method. These three colonies were fixed on a nylonfilm similarly as above. Based on the amino acids 1-7 of SEQ ID NO:7revealed in Experiment 4, an oligonucleotide with a nucleotide sequence,represented by 5'-AAYTAYGAYTAYTAYGARCC-3'(SEQ ID NO:10, was chemicallysynthesized, and labelled with γ-³² P!ATP and T4 polynucleotide kinaseto obtain a synthesized DNA as a second probe. Among the above threecolonies fixed on the nylon film, one colony strongly hybridized withthe second probe was selected by applying conventionalcolony-hybridization method and named "TTP4" as a transformant.

The transformant TTP4 was in a conventional manner inoculated intoL-broth (pH 7.0) containing 100 μg/ml of sodium salt of ampicillin, andincubated at 37° C. for 24 hours under rotary-shaking conditions. Aftercompletion of the culture, the culture was centrifuged to obtain cellswhich were then treated with conventional alkali-SDS method to extract arecombinant DNA. Conventional dideoxy analysis of the recombinant DNArevealed that it contained a DNA with the nucleotide sequence of SEQ IDNO:8 consisting of 3,345 base pairs derived from Thermoanaeroblumbrockii, ATCC 35047. As shown in SEQ ID NO:8, it was revealed that anucleotide sequence, consisting of the bases 596-2,917 in SEQ ID NO:8,encodes an amino acid sequence consisting of 774 amino acids. Comparingthe amino acid sequence deduced from the nucleotide sequence and theN-terminal and internal amino acid sequences of the present trehalosephosphorylase confirmed in Experiment 4, i.e., SEQ ID NOs:1-3 and SEQ IDNOs:6-7, SEQ ID NOs:1-3 were respectively agreed with the amino acids2-6, 308-312, and 633-637 in SEQ ID NO:8, while SEQ ID NOs:6-7 wererespectively agreed with the amino acids 2-31 and 633-647 in SEQ IDNO:8.

These data indicate that the present trehalose phosphorylase has theamino acid sequence of SEQ ID NO:4, and the enzyme of Thermoanaerobiumbrockii, ATCC 35047, is encoded by a DNA having the nucleotide sequenceof SEQ ID NO:5. The recombinant DNA, which was obtained by the abovemethods and revealed its nucleotide sequence, was named "pTTP4". Asshown in FIG.5, the recombinant DNA is positioned at the down stream ofthe recognition site by a restriction enzyme, Pst I.

EXAMPLE A-4

Production of trehalose phosphorylase by transformant

One hundred ml of an aqueous solution, containing 16 g/l polypeptone, 10g/l yeast extract, and 5 g/l sodium chloride, was placed in a 500-mlErlenmeyer flask, autoclaved at 121° C. for 15 min, cooled, asepticallyadjusted to pH 7.0, and aseptically admixed with 10 mg sodium salt ofampicillin into a liquid nutrient medium. The transformant TTP4 inExample A-3 was inoculated into the medium, and incubated at 37° C. forabout 20 hours under aeration-agitation conditions to obtain a seedculture. According to the preparation of the seed culture, 7 L of afresh preparation of the same nutrient culture medium as used in theseed culture was placed in a 10-l fermenter, inoculated with 70 ml ofthe seed culture, and subjected to an incubation for about 20 hoursunder aeration-agitation conditions. The resulting culture was in aconventional manner centrifuged to collect cells which were thensuspended in 10 mM phosphate buffer (pH 7.0), ultrasonicated for-celldisruption, and centrifuged to remove insoluble substances, followed bycollecting a supernatant. The supernatant was dialyzed against 10 mMphosphate buffer, and assayed for trehalose phosphorylase activity inthe supernatant, revealing that about 700 units/l culture of the enzymewas produced.

As a first control, a seed of Escherichia coli XL1-Blue strain wasinoculated into a nutrient culture medium similarly as in the culture ofthe above transformant except that the ampicillin was not added to theculture medium, and cultured. The proliferated cells were disrupted,followed by collecting and dialyzing the supernatant. As a secondcontrol, according to the method in Experiment 1, a seed ofThermoanaerobium brockii, ATCC 35047, was stationary cultured at 60° C.in a nutrient culture medium consisting of the same ingredients as usedin the culture for the transformant except that the ampicillin was notused, and similarly as in the case of the transformant, the cells in theculture were disrupted, followed by collecting and dialyzing thesupernatant. No enzyme activity of the present enzyme was detected inthe dialyzed solution as the first control. The dialyzed solution as thesecond control had an enzyme activity of about 2 units/l culture whichwas lower than that of the transformant TTP4.

In accordance with the method in Experiment 2, the dialyzed solution inExample A-4 was purified on column chromatographies using"DEAE-TOYOPEARL® 650 GEL" and "ULTROGEL® AcA44 RESIN", and the purifiedenzyme was analyzed in accordance with the method in Experiment 3,revealing that it had a molecular weight of 88,000±5,000 daltons onSDS-PAGE, molecular weight of 190,000±10,000 daltons on gel filtrationchromatography, isoelectric point of 5.4±0.5 on electrofocusing usingpolyacrylamide gel, optimum temperature of about 70° C., optimum pH ofabout 7.0-7.5, thermal stability of up to 60° C., and pH stability ofabout 6.0-9.0, all of which were substantially the same as those of theenzyme prepared in Experiments 1 and 2. These results indicate that thepresent trehalose phosphorylase can be sufficiently produced byrecombinant DNA technology, and the enzyme yield can be significantlyincreased thereby.

EXAMPLE A-5

Enzyme solution

The transformant TTP4 in Example A-3 was cultured in a nutrient culturemedium by the method in Example A-4. Cells obtained by centrifuging theculture were disrupted by ultrasonics, and a supernatant of thedisrupted cell suspension was measured for trehalose phosphorylaseactivity. The activity was about 0.7 unit/ml culture. The supernatantwas concentrated with an ultrafiltration membrane, and the concentratewas dialyzed to obtain an enzyme solution with an activity of about 10units/ml of trehalose phosphorylase in a yield of about 70% to the totalenzyme activity of the culture.

EXAMPLE A-6

Saccharide solution containing trehalose

To 25 mM of dipotassium hydrogenphosphate-citric acid buffer (pH 6.0)containing 5% maltose were added 5 units/g maltose of a commerciallyavailable bacterial maltose phosphorylase, and 50 units/g maltose oftrehalose phosphorylase obtained by the method in Example A-1, followedby the incubation at 30° C. for 120 hours. The reaction mixture washeated at 100° C. for 30 min to inactivate the remaining enzymes,cooled, decolored with activated charcoals in a conventional manner,filtered, desalted and purified with ion exchange resins in H- andOH-form, and further concentrated to obtain a 75% syrupy saccharidesolution containing trehalose in a yield of about 95% to the material,d.s.b.

The product, which contains about 45% trehalose, d.s.b., and has asatisfactory sweetness and an adequate viscosity and humectancy, can bearbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies as a sweetener, taste-improving agent, stabilizer,growth-promoting agent for bifid bacteria, andmineral-absorption-promoting agent.

EXAMPLE A-7

Trehalose enriched Powder

A saccharide solution, as a material, containing about 45% trehalose,d.s.b., obtained by the reaction and purification in Example A-6, wasadjusted to give a concentration of about 20%, d.s.b., which was thenmixed with 5 units/g dry solid of glucoamylase and incubated at pH 4.5and 40° C. for 16 hours to decompose the remaining maltose. The reactionmixture was heated at 100° C. for 30 min to suspend the enzymaticreaction, then concentrated into an about 40% solution. To increase thetrehalose content, the concentrated solution was fractionated byproviding four jacketed-stainless steel columns, 3 cm in diameter andone meter in length each, packed with a water suspension of "XT-1016",an alkali metal strong-acid cation exchange resin, Na-form,polymerization degree of 4%, commercialized by Tokyo Organic ChemicalIndustries, Ltd., Tokyo, Japan, which were cascaded in series to give atotal gel-bed depth of about 4 m, feeding 5 v/v % of the solution to theresin, fractionating the solution by feeding water heated to 40° C. tothe columns at SV 0.15, and collecting the resulting trehalose richfractions. The fractions were pooled, concentrated, dried in vacuo, andpulverized to obtain a trehalose rich powder in a yield of about 40% tothe material, d.s.b.

The product contains about 95% trehalose, d.s.b., and has asatisfactorily tastable sweetness and adequate humectancy, and it can bearbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies as a sweetener, taste-improving agent, stabilizer,growth-promoting agent for bifid bacteria, andmineral-absorption-promoting agent.

EXAMPLE A-8

Saccharide solution containing glucosyl-D-galactoside

An aqueous solution containing 5% trehalose, 5% D-galactose, and 5 mMsodium dihydrogenphosphate was adjusted to give a pH of 5.0, admixedwith 10 units/g trehalose of a trehalose phosphorylase obtained by themethod in Example A-1, and enzymatically reacted at 60° C. for 72 hours.The reaction mixture was heated at 90° C. for 30 min to inactivate theremaining enzyme, cooled, decolored with activated charcoals in aconventional manner, filtered, desalted and purified with ion exchangeresins in H- and OH-form, and further concentrated to obtain an about75% syrupy saccharide solution containing glucosylsorbose in a yield ofabout 95% to the material, d.s.b.

The product contains about 22% glucosyl-D-galactoside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies as a sweetener, taste-improving agent, stabilizer,growth-promoting agent for bifid bacteria, andmineral-absorption-promoting agent.

EXAMPLE A-9

Saccharide solution containing glucosyl-D-Qalactoside

An aqueous solution, containing 10% trehalose, 5% D-galactose, and 5 mMsodium dihydrogenphosphate, was adjusted to give a pH of 6.0, admixedwith 30 units/g trehalose of a trehalose phosphorylase obtained by themethod in Example A-1, and enzymatically reacted at 60° C. for 96 hours.The reaction mixture was heated at 90° C. for 30 min to inactivate theremaining enzyme and cooled. Thereafter, 5% by wet weight ofcommercially available baker's yeasts was added to the resulting mixtureto assimilate D-glucose in the reaction mixture while controlling the pHat 5-6 by the addition of 1-N sodium chloride solution and keeping thereaction temperature at 27° C. for 6 hours. The reaction mixture wascentrifuged to remove the yeasts, and the resulting supernatant was in aconventional manner decolored with activated charcoals, filtered,desalted and purified with ion exchangers in H- and OH-form, andconcentrated to obtain a 75% syrup, d.s.b., in a yield of about 65% tothe material, d.s.b.

The product contains about 40% glucosyl-D-galactoside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies as a sweetener, taste-improving agent, stabilizer,growth-promoting agent for bifid bacteria, andmineral-absorption-promoting agent.

EXAMPLE A-10

Glucosyl-D-galactoside rich powder

A saccharide solution, as a material, containing about 22%glucosyl-D-galactoside, d. s.b., obtained by the reaction andpurification of Example A-8, was adjusted to give a concentration ofabout 45%, d.s.b. To increase the content of glucosyl-D-galactoside, theresulting solution was fractionated by providing four jacketed-stainlesssteel columns, 3 cm in diameter and one meter in length each, which werepacked with a water suspension of "XT-1016", an alkali metal strong-acidcation exchange resin, Na-form, polymerization degree of 4%,commercialized by Tokyo Organic Chemical Industries, Ltd., Tokyo, Japan,and cascaded in series to give a total gel-bed depth of about 4 m,feeding 5 v/v % of the solution to the resin, fractionating the solutionby feeding water heated to 40° C. to the columns at SV 0.15 whilekeeping the inner column temperature of 40° C., and collecting theresulting glucosyl-D-galactoside rich fractions. The fractions werepooled, concentrated, dried in vacuo, and pulverized to obtain aglucosyl-D-galactoside rich powder in a yield of about 25% to thematerial, d.s.b.

The product contains about 70% glucosyl-D-galactoside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies as a sweetener, taste-improving agent, stabilizer,growth-promoting agent for bifid bacteria, andmineral-absorption-promoting agent.

EXAMPLE A-11

Saccharide solution containing alucosyl-D-xyloside

An aqueous solution containing 5% trehalose, 2.5% D-xylose, and 5 mMsodium dihydrogenphosphate was adjusted to a pH of 5.0, admixed with 15units/g trehalose of a trehalose phosphorylase obtained by the method inExample A-1, and enzymatically reacted at 60° C. for 72 hours. Thereaction mixture was heated at 90° C. for 30 min to inactivate theremaining enzyme, cooled, decolored with activated charcoal in aconventional manner, filtered, desalted and purified with ion exchangeresins in H- and OH-form, and further concentrated to obtain an about75% syrup in a yield of about 95% to the material, d.s.b.

The product contains about 20% glucosyl-D-xyloside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies.

EXAMPLE A-12

Glucosyl-D-xyloside rich Powder

A saccharide solution as a material containing about 20%glucosyl-D-xyloside, d.s.b., obtained by the reaction and purificationin Example A-11, was adjusted to give a concentration of about 45%,d.s.b. To increase the content of glucosyl-D-xyloside, the resultingsolution was column chromatographed according to the method in ExampleA-10 except for using "DOWEX 50WX4 (Ca-form)", an alkaline-earth metalstrong-acid cation exchange resin commercialized by The Dow ChemicalCo., Midland, Mich., USA, to collect glucosyl-D-xyloside rich fractions.The fractions were pooled, purified, concentrated, dried in vacuo, andpulverized to obtain a glucosyl-D-xyloside rich powder in a yield ofabout 25%, d.s.b.

The product contains about 60% glucosyl-D-xyloside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies.

EXAMPLE A-13

Saccharide solution containing glucosyl-D-fucoside

An aqueous solution, containing 5% trehalose, 2.5% D-fucose, and 5 mMdisodium hydrogenphosphate, was adjusted to give a pH of 5.0, admixedwith 20 units/g trehalose of a trehalose phosphorylase obtained by themethod in Example A-1, and enzymatically reacted at 60° C. for 72 hours.The reaction mixture was heated at 90° C. for 30 min to inactivate theremaining enzyme, cooled, decolored with activated charcoals in aconventional manner, filtered, desalted and purified with ion exchangeresins in H- and OH-form, and further concentrated to obtain an about75% syrup in a yield of about 95% to the material, d.s.b.

The product contains about 20% glucosyl-D-fucoside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies.

EXAMPLE A-14

Saccharide powder rich in glucosyl-D-fucoside

An aqueous solution, containing about 5% trehalose, 2.5% D-fucose, and 5mM sodium dihydrogenphosphate, was adjusted to give a pH of 5.0, admixedwith 20 units/g trehalose of a trehalose phosphorylase obtained by themethod in Example A-1, and enzymatically reacted at 60° C. for 72 hours.The reaction mixture was heated at 100° C. while keeping the pH toalkaline pHs of over 10, cooled, decolored with activated charcoals in aconventional manner, filtered, desalted and purified with ion exchangeresins in H- and OH-form, and further concentrated to obtain a powdercontaining glucosyl-D-fucoside in a yield of about 60% to the material,d.s.b.

The product contains about 50% glucosyl-D-fucoside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies.

EXAMPLE A-15

Saccharide powder containing glucosyl-L-fucoside

An aqueous solution, containing about 5% trehalose, 2.5% L-fucose, and 5mM sodium dihydrogenphosphate, was adjusted to give a pH of 6.0, admixedwith 15 units/g trehalose of a trehalose phosphorylase obtained by themethod in Example A-1, and enzymatically reacted at 60° C. for 72 hours.The reaction mixture was heated at 90° C. for 30 min to inactivate theremaining enzyme, cooled, decolored with activated charcoals in aconventional manner, filtered, desalted and purified with ion exchangeresins in H- and OH-form, and further concentrated to obtain a powdercontaining glucosyl-L-fucoside in a yield of about 95% to the material,d.s.b.

The product contains about 20% glucosyl-L-fucoside, d.s.b., and has ahigh-quality sweetness and adequate humectancy, and it can bearbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies.

EXAMPLE A-16

Saccharide solution containing trehalose

To 25 mM dipotassium hydrogenphosphate-citric acid buffer (pH 6.0)containing 5% maltose were added 5 units/g maltose of a commerciallyavailable maltose phosphorylase and 50 units/g maltose of a trehalosephosphorylase preparation obtained by the method in Example A-5, andsubjected to an enzymatic reaction at 30° C. for 120 hours. The reactionmixture was heated at 100° C. for 30 min to inactivate the remainingenzymes, cooled, decolored with activated charcoals in a conventionalmanner, filtered, desalted and purified with ion exchange resins in H-and OH-form, and further concentrated to obtain an about 75% syrup,d.s.b., in a yield of about 95% to the material, d.s.b.

The product contains about 45% trehalose, d.s.b., and has a high-qualitysweetness and adequate viscosity and humectancy, and it can bearbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies as a sweetener, taste-improving agent, stabilizer,growth-promoting agent for bifid bacteria, andmineral-absorption-promoting agent.

EXAMPLE A-17

Saccharide solution containing glucosyl-D-fucoside

An aqueous solution, containing 5% trehalose, 2.5% D-fucose, and 5 mMdisodium hydrogenphosphate, was adjusted to give a pH of 5.0, admixedwith 20 units/g trehalose of a trehalose phosphorylase obtained by themethod in Example A-5, and enzymatically reacted at 60° C. for 72 hours.The reaction mixture was heated at 90° C. for 30 min to inactivate theremaining enzyme, cooled, decolored with activated charcoals in aconventional manner, filtered, desalted and purified with ion exchangeresins in H- and OH-form, and further concentrated to obtain an about75% syrup in a yield of about 95% to the material, d.s.b.

The product contains about 20% glucosyl-D-fucoside, d.s.b., and has ahigh-quality sweetness and adequate viscosity and humectancy, and it canbe arbitrarily used in food products, cosmetics, pharmaceuticals, andshaped bodies.

The following Example B explains the present saccharide compositionscontaining glucosyl-transferred saccharides:

EXAMPLE B-1

Sweetener

To one part by weight of a glucosyl-D-galactoside rich powder, obtainedby the method in Example A-10, was added 0.05 part by weight of "αGSWEET", an α-glycosyl stevioside commercialized by Toyo Sugar RefiningCo., Ltd., Tokyo, Japan, and the mixture was mixed to homogeneity into apowdery sweetener. The product is a high-quality sweetener with an abouttwofold-higher sweetening power of sucrose and a half calorific value ofsucrose with respect to the sweetening powder. Therefore, the productcan be satisfactorily used as a low-calorie sweetener to sweetenlow-calorie food products for persons, who are restricted to take lesscalories, such as fat persons and diabetics. Since the product lessproduces insoluble glucans and acids by dental caries-inducingmicroorganisms, it can be suitably used to sweeten dental-cariesinhibitory food products.

EXAMPLE B-2

Hard candy

Thirty parts by weight of a saccharide solution containingglucosyl-D-galactoside, obtained by the method in Example A-9, was addedto and dissolved by mixing in 80 parts by weight of hydrogenated maltsyrup with a moisture content of 25%, and the resulting solution wasconcentrated up to give a moisture content of below 2% under reducedpressures, kneaded with one part by weight of citric acid and adequateamounts of a lemon flavor and coloring agent, followed by kneading andshaping the mixture into a hard candy. The product has a high-qualitysweetness, lower humectancy, and satisfactory biting property withoutcausing melting.

EXAMPLE B-3

Chewing gum

Four parts by weight of a powder rich in glucosyl-D-xyloside, obtainedby the method in Example A-12, was admixed with 3 parts by weight ofglucose and 2 parts by weight of a gum base which had been melted byheating until softened, and further mixed with an adequate amount of amint flavor, followed by shaping the mixture by kneading with a rollinto a chewing gum. The product has a satisfactory texture and flavor.

EXAMPLE B-4

Chocolate

Fifteen parts by weight of a powder rich in glucosyl-D-fucoside,obtained by the method in Example A-14, was mixed with 40 parts byweight of cacao paste, 10 parts by weight of cacao butter, 10 parts byweight of sucrose, and 15 parts by weight of skim milk, and the mixturewas passed through a refiner to lower the granular size. Thereafter, theresulting mixture was placed in a conche, mixed with 0.5 part by weightof lecithin, and kneaded up at 50° C. for two days and nights. Thekneaded mixture was poured into a molding machine, shaped, andsolidified into a chocolate. The product free of fat- and sugar-bloomshas a satisfactory taste, flavor, and meltability on your tongue.

EXAMPLE B-5

Custard cream

To 400 parts by weight of a powder rich in glucosyl-L-fucoside, obtainedby the method in Example A-15, were added 500 parts by weight of cornstarch, 500 parts by weight of maltose, and 5 parts by weight of salt,and the mixture was sufficiently mixed by passing through a sieve, mixedwith 1,400 parts by weight of fresh eggs, stirred, gradually admixedwith 5,000 parts by weight of a boiling milk, and heated over a slowfire while stirring. The heating was suspended when the corn starchcompletely gelatinized to show semitransparency, then cooled, mixed witha small amount of a vanilla flavor to obtain a custard cream. Theproduct has a smooth surface and satisfactory taste free of strongsweetness.

EXAMPLE B-6

Uiro (starch paste)

To 90 parts by weight of a saccharide solution containingglucosyl-D-fucoside, obtained by the method in Example A-13, were added90 parts by weight of rice powder, 20 parts by weight of corn starch, 20parts by weight of sugar, one part by weight of matcha (a green tee)powder, and an adequate amount of water, and the mixture was kneaded,placed in a container, and steamed for 60 min into a matcha uiro. Theproduct has a satisfactory gloss, biting property, flavor, and taste.The retrogradation of starch is well prevented, resulting in arelatively-long shelf life.

EXAMPLE B-7

Bettara-zuke (fresh radish pickles)

A premix for bettara-zuke was prepared by mixing to homogeneity one partby weight of a saccharide solution containing glucosyl-D-galactoside,obtained by the method in Example A-8, with 3 parts by weight ofmaltose, 0.05 part by weight of a licorice preparation, 0.008 part byweight of malic acid, 0.07 part by weight of sodium glutamate, 0.03 partby weight of potassium sorbate, and 0.2 part by weight of pullulan.Thirty kilograms of radish was first pickled with salt in a conventionalmanner, then pickled with sugar, and soaked in a seasoning solution,prepared with 4 kg of the premix, into the desired product. The producthas a satisfactory color, gloss, and fragrance, as well as an adequatesweetness, and satisfactory biting property.

EXAMPLE B-8

Beverage with lactic acid bacteria

One hundred and thirty parts by weight of a saccharide solutioncontaining glucosyl-D-xyloside, obtained by the method in Example A-11,175 parts by weight of skim milk, and 50 parts by weight of"NYUKAOLIGO®", a high lactosucrose content powder commercialized byHayashibara Shoji, Inc., Okayama, Japan, were dissolved in 1,150 partsby weight of water, and the solution was sterilized at 65° C. for 30min, cooled to 40° C., and in a conventional manner inoculated with 30parts by weight of lactic acid bacteria as a starter, followed by theincubation at 37° C. for 8 hours to obtain the desired product. Theproduct is a beverage containing lactic acid bacteria and having asatisfactory flavor and taste. The product contains oligosaccharideswhich stabilize the bacteria and promote the growth.

EXAMPLE B-9

Skin cream

To 4 parts by weight of a powder rich in trehalose, obtained by themethod in Example A-7, were added 2 parts by weight of polyoxyethyleneglycol monostearate, 5 parts by weight of self-emulsifying glycerinemonostearate, 2 parts by weight of α-glycosyl rutin, one part by weightof liquid paraffin, 10 parts by weight of glycerol trioctanate, and anappropriate amount of an antiseptic, and the mixture was dissolved byheating in a conventional manner, mixed with 5 parts by weight of1,3-butylene glycol, and 66 parts by weight of refined water, emulsifiedwith a homogenizer, and mixed with an appropriate amount of a flavorinto a skin cream. The product with a well-spreadability can bearbitrarily used as a sunscreen, skin-refining agent, and skin-whiteningagent.

EXAMPLE B-10

Toothpaste

Forty-five parts by weight of calcium hydrogen phosphate, 1.5 parts byweight of sodium lauryl sulfate, 25 parts by weight of glycerine, 0.5part by weight of polyoxyethylene sorbitan laurate, 0.02 part by weightof saccharin, 0.05 part by weight of an antiseptic, and 13 parts byweight of water were mixed with 15 parts by weight of a saccharidesolution rich in trehalose, obtained by the method in Example A-6, intoa toothpaste.

The product, having a superior gloss and detergency, can be suitablyused as a dentifrice.

EXAMPLE B-11

Nutrition for intubation feeding

A composition consisting of the following ingredients was prepared: 80parts by weight of a powder rich in glucosyl-D-galactoside obtained bythe method in Experiment 8, 190 parts by weight of dried egg yolk, 209parts by weight of skim milk, 4.4 parts by weight of sodium chloride,1.85 parts by weight of potassium chloride, 4 parts by weight ofmagnesium sulfate, 0.01 part by weight of thiamine, and 0.1 part byweight of sodium ascorbate, 0.6 part by weight of vitamin E acetate, and0.04 part by weight of nicotine amide. Twenty-five grams aliquots of thecomposition were injected into small laminated aluminum bags which werethen heat-sealed to obtain the desired product.

One bag of the product is dissolved in about 150-300 ml water into asupplemental nutrition feeding before administering to the nasal cavity,throat, or stomach.

EXAMPLE B-12

Strawberry jam

One hundred and fifty parts by weight of fresh strawberries, 60 parts byweight of sucrose, 20 parts by weight of maltose, 40 parts by weight ofa saccharide solution containing trehalose obtained by the method inExample A-16, 5 parts by weight of pectin, and one part by weight ofcitric acid. The mixture was boiled up in a pan and bottled into thedesired product. The product has a satisfactory taste, flavor, andcolor.

EXAMPLE B-13

Sweetened condensed milk

In 100 parts by weight of fresh milk were dissolved one part by weightof sucrose and 3 parts by weight of a saccharide solution containingglucosyl-D-fucoside obtained by the method in Example A-17, and thesolution was sterilized by heating on a plate heater, condensed to givea concentration of about 70%, and aseptically canned into the desiredproduct. The product has a mild sweetness, flavor, and taste, and it canbe arbitrarily used as a seasoning for food for infants, fruits,coffees, cocoas, and teas.

EFFECT OF THE INVENTION

As evident from the above, the present invention was made based on afinding of a novel trehalose phosphorylase which has a higher optimumtemperature and thermal stability than those of conventional trehalosephosphorylases. The trehalose phosphorylase according to the presentinvention has a relatively-wide range pH-stability in which the optimumpH lies. The trehalose phosphorylase can be produced by microorganismscapable of producing the enzyme in a satisfactorily-high yield. Thus,when the present trehalose phosphorylase is allowed to contact withβ-D-glucose-1-phosphoric acid as a saccharide donor in the presence ofother saccharides, glucosyl-transferred saccharides includingglucosyl-D-galactoside, which are conventionally known but scarcelyobtainable, can be produced on an industrial-scale and in arelatively-low cost.

The glucosyl-transferred saccharides and saccharide compositionscontaining the same can be used as sweeteners with a relatively-highquality sweetness, taste-improving agents, quality-improving agents,body-imparting agents, viscosity-controlling agents,moisture-controlling agents, gloss-imparting agents, and supplementalnutrition agents in food products, cosmetics, pharmaceuticals, andshaped bodies. Because of these outstanding characteristics of thepresent invention, it greatly contributes to food, cosmetic, andpharmaceutical fields, and to agriculture, fishery, breeding, andchemical industries.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 10    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:5 amino acid - #s              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:peptide    -      (v) FRAGMENT TYPE:N-terminal fragment    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1:    - Ala Asn Lys Thr Lys    1               5    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:5 amino acid - #s              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:peptide    -      (v) FRAGMENT TYPE:internal fragment    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2:    - Glu Gln Glu Glu Phe    1               5    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:5 amino acid - #s              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:peptide    -      (v) FRAGMENT TYPE:internal fragment    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:3:    - Asn Tyr Asp Tyr Tyr    1               5    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:773 amino ac - #ids              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:peptide    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:4:    - Ala Asn Lys Thr Lys Lys Pro Ile Tyr Pro Ph - #e Glu Asp Trp Val Ile    #                15    - Arg Glu Thr Gln Phe Ser Ile Asp Thr Asn Ty - #r Arg Asn Glu Thr Ile    #            30    - Phe Thr Leu Ala Asn Gly Tyr Ile Gly Met Ar - #g Gly Thr Phe Glu Glu    #        45    - Arg Tyr Ser Gly Pro Lys Asn Thr Ser Phe As - #n Gly Thr Tyr Ile Asn    #    60    - Gly Phe Tyr Glu Ile His Asp Ile Val Tyr Pr - #o Glu Gly Gly Tyr Gly    #80    - Phe Ala Lys Ile Gly Gln Thr Met Leu Asn Va - #l Ala Asp Ser Lys Ile    #                95    - Ile Lys Leu Tyr Val Asp Gly Glu Glu Phe As - #p Leu Leu Gln Gly Lys    #           110    - Ile Leu Phe Tyr Glu Arg Val Leu Asp Met Ly - #s Lys Gly Phe Val Glu    #       125    - Arg Lys Val Lys Trp Glu Ser Pro Thr Gly Ly - #s Ile Leu Glu Val Lys    #   140    - Ile Lys Arg Ile Val Ser Leu Asn Arg Gln Hi - #s Leu Ala Ala Ile Ser    145                 1 - #50                 1 - #55                 1 -    #60    - Phe Thr Met Gln Pro Val Asn Phe Thr Gly Ly - #s Ile Arg Phe Val Ser    #               175    - Ala Ile Asp Gly Asn Val Ser Asn Ile Asn As - #p Ser Glu Asp Val Arg    #           190    - Val Gly Ser Asn Leu Lys Gly Lys Val Leu Ly - #s Thr Ile Asp Lys Ser    #       205    - Val Glu Gly Leu Lys Gly Trp Ile Val Gln Ly - #s Thr Gln Lys Ser Asn    #   220    - Phe Ser Tyr Ala Cys Ala Ile Asp Asn Val Le - #u Val Ala Asp Ser Lys    225                 2 - #30                 2 - #35                 2 -    #40    - Tyr Glu Val Ser Asn Ser Leu Glu Glu Asp Gl - #y Val Lys Val Ile Val    #               255    - Asp Leu Glu Ala Glu Lys Gly Thr Ser Tyr Th - #r Leu Asn Lys Phe Ile    #           270    - Ser Tyr Tyr Thr Ser Lys Asp Phe Asp Glu As - #n Lys Leu Val Ala Leu    #       285    - Ala Leu Glu Glu Ile Glu Lys Ala Lys Asn As - #p Gly Phe Glu Thr Ile    #   300    - Glu Lys Glu Gln Glu Glu Phe Leu Asn Ser Ph - #e Trp Lys Asp Ala Asp    305                 3 - #10                 3 - #15                 3 -    #20    - Val Ile Ile Glu Gly Asp Lys Ala Leu Gln Gl - #n Gly Ile Arg Phe Asn    #               335    - Glu Phe His Leu Leu Gln Ser Val Gly Arg As - #p Gly Lys Thr Asn Ile    #           350    - Ala Ala Lys Gly Leu Thr Gly Gly Gly Tyr Gl - #u Gly His Tyr Phe Trp    #   365    - Asp Ser Asp Ile Tyr Ile Met Pro Phe Phe Le - #u Tyr Thr Lys Pro Glu    #   380    - Ile Ala Lys Ala Leu Val Met Tyr Arg Tyr As - #n Leu Leu Asp Ala Ala    385                 3 - #90                 3 - #95                 4 -    #00    - Arg Ser Arg Ala Lys Glu Leu Gly His Lys Gl - #y Ala Leu Tyr Pro Trp    #               415    - Arg Thr Ile Asp Gly Pro Glu Cys Ser Ala Ty - #r Phe Pro Ala Gly Thr    #           430    - Ala Gln Tyr His Ile Asn Ala Asp Ile Val Ty - #r Ala Leu Lys Arg Tyr    #       445    - Val Glu Ala Thr Asn Asp Val Asp Phe Leu Ty - #r Asp Tyr Gly Cys Glu    #   460    - Ile Leu Phe Glu Thr Ala Arg Phe Trp Glu As - #p Leu Gly Ala Tyr Ile    465                 4 - #70                 4 - #75                 4 -    #80    - Pro Leu Lys Gly Asn Lys Phe Cys Ile Asn Cy - #s Val Thr Gly Pro Asp    #               495    - Glu Tyr Thr Ala Leu Val Asp Asn Asn Ala Ty - #r Thr Asn Tyr Met Ala    #           510    - Lys Met Asn Leu Glu Tyr Ala Tyr Asp Ile Al - #a Asn Lys Met Lys Lys    #       525    - Glu Val Pro Gln Lys Tyr Gln Lys Val Ala Se - #r Lys Leu Asn Leu Lys    #   540    - Asp Glu Glu Ile Val Ala Trp Lys Lys Ala Al - #a Asp Asn Met Tyr Leu    545                 5 - #50                 5 - #55                 5 -    #60    - Pro Tyr Ser Lys Glu Leu Asp Ile Ile Pro Gl - #n Asp Asp Ser Phe Leu    #               575    - Tyr Lys Glu Arg Ile Thr Val Asp Glu Ile Pr - #o Glu Asp Gln Phe Pro    #           590    - Leu Leu Leu His Trp His Tyr Leu Asn Ile Ty - #r Arg Tyr Gln Ile Cys    #       605    - Lys Gln Pro Asp Val Leu Leu Leu Met Phe Le - #u Gln Arg Glu Lys Phe    #   620    - Thr Lys Asp Glu Leu Lys Lys Asn Tyr Asp Ty - #r Tyr Glu Pro Ile Thr    625                 6 - #30                 6 - #35                 6 -    #40    - Thr His Asp Ser Ser Leu Ser Pro Ala Ile Ph - #e Ser Ile Leu Ala Asn    #               655    - Glu Ile Gly Tyr Thr Asp Lys Ala Tyr Lys Ty - #r Phe Met Met Thr Ala    #           670    - Arg Met Asp Leu Asp Asp Tyr Asn Asp Asn Va - #l Lys Asp Gly Ile His    #       685    - Ala Ala Ser Met Ala Gly Thr Trp Ser Ala Va - #l Val Asn Gly Phe Gly    #   700    - Gly Met Arg Val Tyr Thr Asn Glu Leu His Ph - #e Glu Pro Arg Leu Pro    705                 7 - #10                 7 - #15                 7 -    #20    - Lys Glu Trp Asn Leu Leu Ser Phe Asn Val Ar - #g Tyr Lys Gly Arg Lys    #               735    - Ile Asn Val Lys Leu Thr Lys Glu Asn Val Va - #l Phe Ala Leu Leu Glu    #           750    - Gly Glu Pro Ile Glu Ile Tyr Tyr Phe Asp Ly - #s Lys Ile Leu Leu Glu    #       765    - Lys Gly Glu Ile Lys        770    - (2) INFORMATION FOR SEQ ID NO:5:    -       (i) SEQUENCE CHARACTERISTICS:    -           (A) LENGTH:2319 base pa - #irs              (B) TYPE:nucleic acid              (C) STRANDEDNESS:double              (D) TOPOLOGY:linear    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:5:    - GCCAACAAAA CGAAGAAACC AATTTACCCT TTTGAAGATT GGGTTATAAG AG - #AGACGCAG    60    - TTTAGTATAG ATACTAACTA TAGAAATGAA ACTATTTTTA CTTTAGCAAA TG - #GATATATT    120    - GGAATGAGAG GAACTTTTGA GGAAAGATAT TCAGGGCCTA AAAATACTTC TT - #TTAATGGG    180    - ACGTATATCA ATGGGTTTTA TGAAATACAC GATATAGTTT ACCCTGAAGG GG - #GATATGGT    240    - TTTGCAAAAA TAGGGCAGAC GATGCTAAAT GTTGCTGATA GCAAAATAAT AA - #AATTATAT    300    - GTAGATGGGG AAGAGTTTGA TTTGTTACAA GGGAAAATCC TCTTTTATGA GA - #GAGTACTT    360    - GACATGAAGA AAGGTTTTGT AGAAAGAAAA GTAAAATGGG AATCCCCTAC AG - #GAAAAATT    420    - TTAGAGGTAA AAATAAAGAG AATTGTATCA TTAAATAGAC AACATTTAGC GG - #CGATTTCT    480    - TTTACTATGC AACCTGTAAA TTTTACCGGA AAAATTAGAT TTGTTTCCGC TA - #TTGACGGA    540    - AATGTTTCAA ATATAAATGA TAGTGAAGAT GTAAGAGTAG GGTCAAATTT AA - #AAGGAAAG    600    - GTTTTAAAGA CTATAGATAA AAGTGTAGAG GGTTTAAAAG GGTGGATTGT TC - #AAAAGACA    660    - CAAAAGAGCA ATTTCTCCTA TGCTTGCGCG ATAGACAATG TATTAGTGGC AG - #ATAGCAAA    720    - TATGAAGTCT CAAATAGTTT AGAAGAAGAT GGAGTAAAAG TAATTGTAGA TC - #TAGAGGCT    780    - GAAAAAGGCA CCTCATACAC TTTGAATAAA TTTATTTCCT ATTACACTTC AA - #AGGATTTT    840    - GATGAAAATA AATTGGTTGC TCTTGCTTTA GAAGAAATAG AAAAAGCCAA AA - #ATGACGGC    900    - TTTGAAACGA TAGAAAAAGA GCAGGAAGAA TTTTTGAATT CTTTTTGGAA AG - #ATGCTGAT    960    - GTAATCATAG AAGGAGATAA AGCTCTGCAG CAAGGCATAC GCTTTAATGA AT - #TTCATCTA    1020    - CTTCAATCTG TCGGAAGAGA TGGAAAGACA AATATTGCAG CAAAAGGGCT GA - #CTGGAGGA    1080    - GGTTATGAAG GCCATTATTT TTGGGATTCT GATATCTATA TAATGCCTTT CT - #TTCTTTAT    1140    - ACAAAGCCTG AAATTGCAAA AGCTTTGGTA ATGTACAGGT ATAATCTTTT GG - #ATGCAGCA    1200    - AGATCCAGGG CAAAGGAATT AGGTCACAAA GGAGCTTTGT ATCCTTGGAG AA - #CGATAGAT    1260    - GGTCCTGAAT GTTCTGCTTA CTTTCCAGCT GGTACGGCAC AGTATCACAT AA - #ATGCTGAT    1320    - ATAGTTTATG CTTTGAAAAG ATATGTAGAA GCGACGAATG ACGTGGATTT TC - #TTTATGAC    1380    - TACGGTTGTG AAATATTATT TGAAACTGCA AGATTTTGGG AAGATTTAGG AG - #CGTATATT    1440    - CCTCTTAAGG GCAATAAATT CTGCATAAAC TGTGTCACTG GTCCGGATGA GT - #ATACGGCA    1500    - TTAGTTGACA ATAACGCTTA TACCAATTAT ATGGCGAAAA TGAATTTGGA AT - #ATGCCTAT    1560    - GACATTGCAA ACAAAATGAA AAAAGAAGTG CCTCAAAAAT ATCAAAAAGT CG - #CTTCTAAA    1620    - CTAAATCTAA AGGATGAAGA AATTGTTGCG TGGAAAAAAG CTGCTGACAA TA - #TGTACCTT    1680    - CCTTATTCAA AAGAACTTGA TATTATACCA CAGGATGACA GTTTTTTGTA TA - #AAGAAAGG    1740    - ATAACAGTGG ATGAAATACC TGAGGACCAA TTTCCACTTT TATTGCACTG GC - #ATTACCTA    1800    - AATATTTACA GATATCAAAT ATGCAAACAG CCTGATGTGT TGCTTTTGAT GT - #TTTTACAG    1860    - AGAGAAAAAT TTACTAAAGA TGAACTTAAA AAGAATTACG ATTATTATGA AC - #CTATTACC    1920    - ACTCACGACT CCTCCTTGTC GCCAGCTATA TTTAGCATAC TAGCCAATGA AA - #TAGGATAT    1980    - ACTGACAAGG CTTATAAATA CTTTATGATG ACTGCAAGAA TGGATTTGGA TG - #ACTACAAT    2040    - GACAATGTTA AGGACGGAAT TCACGCTGCT TCTATGGCAG GGACATGGAG CG - #CAGTTGTG    2100    - AATGGTTTTG GTGGAATGAG GGTTTATACA AATGAACTGC ATTTTGAGCC GA - #GATTGCCA    2160    - AAAGAATGGA ATTTGCTCTC TTTTAATGTG AGATACAAAG GGAGAAAAAT AA - #ATGTCAAA    2220    - TTAACCAAAG AAAATGTTGT GTTTGCATTA TTAGAAGGAG AGCCTATAGA AA - #TCTACTAC    2280    #2319              TACT TGAAAAAGGA GAAATAAAG    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:30 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:peptide    -      (v) FRAGMENT TYPE:N-terminal fragment    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:6:    - Ala Asn Lys Thr Lys Lys Pro Ile Tyr Pro Ph - #e Glu Asp Trp Val Ile    #                15    - Arg Glu Thr Gln Phe Ser Ile Asp Thr Asn Ty - #r Arg Asn Glu    #            30    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:15 amino aci - #ds              (B) TYPE:amino acid              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:peptide    -      (v) FRAGMENT TYPE:internal fragment    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:7:    - Asn Tyr Asp Tyr Tyr Glu Pro Ile Thr Thr Hi - #s Asp Ser Ser Leu    #                15    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:3345 base pa - #irs              (B) TYPE:nucleic acid              (C) STRANDEDNESS:double              (D) TOPOLOGY:linear    -     (ii) MOLECULE TYPE:Genomic DNA    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM:Thermoanaerobium - # brockii              (F) STRAIN:ATCC 35047    -     (ix) FEATURE:              (A1)NAME/KEY:1-595 5'-UTR              (C1)IDENTIFICATION METHOD:E    #peptide  (A2)NAME/KEY:596-2917 mat              (C2)IDENTIFICATION METHOD:S              (A3)NAME/KEY:2918-3345 3'- - #UTR              (C3)IDENTIFICATION METHOD:E    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:8:    - CTGACTGGAA TACACCTGTA GAATATCTTG CAAAAGAGAG CGTATATTTG GT - #TCAAAATT    60    - GGCCGTACAC TGCAAACGTT CTTGTAGAGC AGTATGGAAA AAAGAACATT TT - #GGCATATC    120    - ACGGATGGAC AGGTCCGGTT AAAGAGTCCC ACGTTTTGGG AGGAGAAGTT AT - #AGGAATAC    180    - CAACTGGTGC ACCTAATAAA GAGATGGCTA TAAAGTTTAT GGAATACCTT AT - #GAGTAAAG    240    - AAGTTCAAGA GAAACTTGTC ACTAAATTAG GATGGCCATC CATGAGAAGT GA - #CGCTTATG    300    - GGAAGGTTGC AGAGTGGCAA AAACCATATT TTGAAGCTAT AAATGAAGCG TT - #AAAACATG    360    - CAGAACCAAG GCCAAACCTT GTATACTGGG CTGATGTGGA CAAAGCTATA AA - #TGGAGCAT    420    - TGAGAGAAAT AATATTTGAA GGCAAAGATA TCAAGACAAC TCTTGACAAA TA - #TCACAACA    480    - TGATAGAAGA AGCTAAGAAA GCTGCAGAAA GCAAGTAAAT GTTTTAAATT GT - #TTTAGTCG    540    - GAAACGACTT TGTTTCCGAC TAAAATTTTG AATAAAGTAA GAGTGGAGGA TG - #GAT    595    - ATG GCC AAC AAA ACG AAG AAA CCA ATT TAC CC - #T TTT GAA GAT TGG GTT    643    Met Ala Asn Lys Thr Lys Lys Pro Ile Tyr Pr - #o Phe Glu Asp Trp Val    #                15    - ATA AGA GAG ACG CAG TTT AGT ATA GAT ACT AA - #C TAT AGA AAT GAA ACT    691    Ile Arg Glu Thr Gln Phe Ser Ile Asp Thr As - #n Tyr Arg Asn Glu Thr    #            30    - ATT TTT ACT TTA GCA AAT GGA TAT ATT GGA AT - #G AGA GGA ACT TTT GAG    739    Ile Phe Thr Leu Ala Asn Gly Tyr Ile Gly Me - #t Arg Gly Thr Phe Glu    #        45    - GAA AGA TAT TCA GGG CCT AAA AAT ACT TCT TT - #T AAT GGG ACG TAT ATC    787    Glu Arg Tyr Ser Gly Pro Lys Asn Thr Ser Ph - #e Asn Gly Thr Tyr Ile    #    60    - AAT GGG TTT TAT GAA ATA CAC GAT ATA GTT TA - #C CCT GAA GGG GGA TAT    835    Asn Gly Phe Tyr Glu Ile His Asp Ile Val Ty - #r Pro Glu Gly Gly Tyr    #80    - GGT TTT GCA AAA ATA GGG CAG ACG ATG CTA AA - #T GTT GCT GAT AGC AAA    883    Gly Phe Ala Lys Ile Gly Gln Thr Met Leu As - #n Val Ala Asp Ser Lys    #                95    - ATA ATA AAA TTA TAT GTA GAT GGG GAA GAG TT - #T GAT TTG TTA CAA GGG    931    Ile Ile Lys Leu Tyr Val Asp Gly Glu Glu Ph - #e Asp Leu Leu Gln Gly    #           110    - AAA ATC CTC TTT TAT GAG AGA GTA CTT GAC AT - #G AAG AAA GGT TTT GTA    979    Lys Ile Leu Phe Tyr Glu Arg Val Leu Asp Me - #t Lys Lys Gly Phe Val    #       125    - GAA AGA AAA GTA AAA TGG GAA TCC CCT ACA GG - #A AAA ATT TTA GAG GTA    1027    Glu Arg Lys Val Lys Trp Glu Ser Pro Thr Gl - #y Lys Ile Leu Glu Val    #   140    - AAA ATA AAG AGA ATT GTA TCA TTA AAT AGA CA - #A CAT TTA GCG GCG ATT    1075    Lys Ile Lys Arg Ile Val Ser Leu Asn Arg Gl - #n His Leu Ala Ala Ile    145                 1 - #50                 1 - #55                 1 -    #60    - TCT TTT ACT ATG CAA CCT GTA AAT TTT ACC GG - #A AAA ATT AGA TTT GTT    1123    Ser Phe Thr Met Gln Pro Val Asn Phe Thr Gl - #y Lys Ile Arg Phe Val    #               175    - TCC GCT ATT GAC GGA AAT GTT TCA AAT ATA AA - #T GAT AGT GAA GAT GTA    1171    Ser Ala Ile Asp Gly Asn Val Ser Asn Ile As - #n Asp Ser Glu Asp Val    #           190    - AGA GTA GGG TCA AAT TTA AAA GGA AAG GTT TT - #A AAG ACT ATA GAT AAA    1219    Arg Val Gly Ser Asn Leu Lys Gly Lys Val Le - #u Lys Thr Ile Asp Lys    #       205    - AGT GTA GAG GGT TTA AAA GGG TGG ATT GTT CA - #A AAG ACA CAA AAG AGC    1267    Ser Val Glu Gly Leu Lys Gly Trp Ile Val Gl - #n Lys Thr Gln Lys Ser    #   220    - AAT TTC TCC TAT GCT TGC GCG ATA GAC AAT GT - #A TTA GTG GCA GAT AGC    1315    Asn Phe Ser Tyr Ala Cys Ala Ile Asp Asn Va - #l Leu Val Ala Asp Ser    225                 2 - #30                 2 - #35                 2 -    #40    - AAA TAT GAA GTC TCA AAT AGT TTA GAA GAA GA - #T GGA GTA AAA GTA ATT    1363    Lys Tyr Glu Val Ser Asn Ser Leu Glu Glu As - #p Gly Val Lys Val Ile    #               255    - GTA GAT CTA GAG GCT GAA AAA GGC ACC TCA TA - #C ACT TTG AAT AAA TTT    1411    Val Asp Leu Glu Ala Glu Lys Gly Thr Ser Ty - #r Thr Leu Asn Lys Phe    #           270    - ATT TCC TAT TAC ACT TCA AAG GAT TTT GAT GA - #A AAT AAA TTG GTT GCT    1459    Ile Ser Tyr Tyr Thr Ser Lys Asp Phe Asp Gl - #u Asn Lys Leu Val Ala    #       285    - CTT GCT TTA GAA GAA ATA GAA AAA GCC AAA AA - #T GAC GGC TTT GAA ACG    1507    Leu Ala Leu Glu Glu Ile Glu Lys Ala Lys As - #n Asp Gly Phe Glu Thr    #   300    - ATA GAA AAA GAG CAG GAA GAA TTT TTG AAT TC - #T TTT TGG AAA GAT GCT    1555    Ile Glu Lys Glu Gln Glu Glu Phe Leu Asn Se - #r Phe Trp Lys Asp Ala    305                 3 - #10                 3 - #15                 3 -    #20    - GAT GTA ATC ATA GAA GGA GAT AAA GCT CTG CA - #G CAA GGC ATA CGC TTT    1603    Asp Val Ile Ile Glu Gly Asp Lys Ala Leu Gl - #n Gln Gly Ile Arg Phe    #               335    - AAT GAA TTT CAT CTA CTT CAA TCT GTC GGA AG - #A GAT GGA AAG ACA AAT    1651    Asn Glu Phe His Leu Leu Gln Ser Val Gly Ar - #g Asp Gly Lys Thr Asn    #           350    - ATT GCA GCA AAA GGG CTG ACT GGA GGA GGT TA - #T GAA GGC CAT TAT TTT    1699    Ile Ala Ala Lys Gly Leu Thr Gly Gly Gly Ty - #r Glu Gly His Tyr Phe    #       365    - TGG GAT TCT GAT ATC TAT ATA ATG CCT TTC TT - #T CTT TAT ACA AAG CCT    1747    Trp Asp Ser Asp Ile Tyr Ile Met Pro Phe Ph - #e Leu Tyr Thr Lys Pro    #   380    - GAA ATT GCA AAA GCT TTG GTA ATG TAC AGG TA - #T AAT CTT TTG GAT GCA    1795    Glu Ile Ala Lys Ala Leu Val Met Tyr Arg Ty - #r Asn Leu Leu Asp Ala    385                 3 - #90                 3 - #95                 4 -    #00    - GCA AGA TCC AGG GCA AAG GAA TTA GGT CAC AA - #A GGA GCT TTG TAT CCT    1843    Ala Arg Ser Arg Ala Lys Glu Leu Gly His Ly - #s Gly Ala Leu Tyr Pro    #               415    - TGG AGA ACG ATA GAT GGT CCT GAA TGT TCT GC - #T TAC TTT CCA GCT GGT    1891    Trp Arg Thr Ile Asp Gly Pro Glu Cys Ser Al - #a Tyr Phe Pro Ala Gly    #           430    - ACG GCA CAG TAT CAC ATA AAT GCT GAT ATA GT - #T TAT GCT TTG AAA AGA    1939    Thr Ala Gln Tyr His Ile Asn Ala Asp Ile Va - #l Tyr Ala Leu Lys Arg    #       445    - TAT GTA GAA GCG ACG AAT GAC GTG GAT TTT CT - #T TAT GAC TAC GGT TGT    1987    Tyr Val Glu Ala Thr Asn Asp Val Asp Phe Le - #u Tyr Asp Tyr Gly Cys    #   460    - GAA ATA TTA TTT GAA ACT GCA AGA TTT TGG GA - #A GAT TTA GGA GCG TAT    2035    Glu Ile Leu Phe Glu Thr Ala Arg Phe Trp Gl - #u Asp Leu Gly Ala Tyr    465                 4 - #70                 4 - #75                 4 -    #80    - ATT CCT CTT AAG GGC AAT AAA TTC TGC ATA AA - #C TGT GTC ACT GGT CCG    2083    Ile Pro Leu Lys Gly Asn Lys Phe Cys Ile As - #n Cys Val Thr Gly Pro    #               495    - GAT GAG TAT ACG GCA TTA GTT GAC AAT AAC GC - #T TAT ACC AAT TAT ATG    2131    Asp Glu Tyr Thr Ala Leu Val Asp Asn Asn Al - #a Tyr Thr Asn Tyr Met    #           510    - GCG AAA ATG AAT TTG GAA TAT GCC TAT GAC AT - #T GCA AAC AAA ATG AAA    2179    Ala Lys Met Asn Leu Glu Tyr Ala Tyr Asp Il - #e Ala Asn Lys Met Lys    #       525    - AAA GAA GTG CCT CAA AAA TAT CAA AAA GTC GC - #T TCT AAA CTA AAT CTA    2227    Lys Glu Val Pro Gln Lys Tyr Gln Lys Val Al - #a Ser Lys Leu Asn Leu    #   540    - AAG GAT GAA GAA ATT GTT GCG TGG AAA AAA GC - #T GCT GAC AAT ATG TAC    2275    Lys Asp Glu Glu Ile Val Ala Trp Lys Lys Al - #a Ala Asp Asn Met Tyr    545                 5 - #50                 5 - #55                 5 -    #60    - CTT CCT TAT TCA AAA GAA CTT GAT ATT ATA CC - #A CAG GAT GAC AGT TTT    2323    Leu Pro Tyr Ser Lys Glu Leu Asp Ile Ile Pr - #o Gln Asp Asp Ser Phe    #               575    - TTG TAT AAA GAA AGG ATA ACA GTG GAT GAA AT - #A CCT GAG GAC CAA TTT    2371    Leu Tyr Lys Glu Arg Ile Thr Val Asp Glu Il - #e Pro Glu Asp Gln Phe    #           590    - CCA CTT TTA TTG CAC TGG CAT TAC CTA AAT AT - #T TAC AGA TAT CAA ATA    2419    Pro Leu Leu Leu His Trp His Tyr Leu Asn Il - #e Tyr Arg Tyr Gln Ile    #       605    - TGC AAA CAG CCT GAT GTG TTG CTT TTG ATG TT - #T TTA CAG AGA GAA AAA    2467    Cys Lys Gln Pro Asp Val Leu Leu Leu Met Ph - #e Leu Gln Arg Glu Lys    #   620    - TTT ACT AAA GAT GAA CTT AAA AAG AAT TAC GA - #T TAT TAT GAA CCT ATT    2515    Phe Thr Lys Asp Glu Leu Lys Lys Asn Tyr As - #p Tyr Tyr Glu Pro Ile    625                 6 - #30                 6 - #35                 6 -    #40    - ACC ACT CAC GAC TCC TCC TTG TCG CCA GCT AT - #A TTT AGC ATA CTA GCC    2563    Thr Thr His Asp Ser Ser Leu Ser Pro Ala Il - #e Phe Ser Ile Leu Ala    #               655    - AAT GAA ATA GGA TAT ACT GAC AAG GCT TAT AA - #A TAC TTT ATG ATG ACT    2611    Asn Glu Ile Gly Tyr Thr Asp Lys Ala Tyr Ly - #s Tyr Phe Met Met Thr    #           670    - GCA AGA ATG GAT TTG GAT GAC TAC AAT GAC AA - #T GTT AAG GAC GGA ATT    2659    Ala Arg Met Asp Leu Asp Asp Tyr Asn Asp As - #n Val Lys Asp Gly Ile    #       685    - CAC GCT GCT TCT ATG GCA GGG ACA TGG AGC GC - #A GTT GTG AAT GGT TTT    2707    His Ala Ala Ser Met Ala Gly Thr Trp Ser Al - #a Val Val Asn Gly Phe    #   700    - GGT GGA ATG AGG GTT TAT ACA AAT GAA CTG CA - #T TTT GAG CCG AGA TTG    2755    Gly Gly Met Arg Val Tyr Thr Asn Glu Leu Hi - #s Phe Glu Pro Arg Leu    705                 7 - #10                 7 - #15                 7 -    #20    - CCA AAA GAA TGG AAT TTG CTC TCT TTT AAT GT - #G AGA TAC AAA GGG AGA    2803    Pro Lys Glu Trp Asn Leu Leu Ser Phe Asn Va - #l Arg Tyr Lys Gly Arg    #               735    - AAA ATA AAT GTC AAA TTA ACC AAA GAA AAT GT - #T GTG TTT GCA TTA TTA    2851    Lys Ile Asn Val Lys Leu Thr Lys Glu Asn Va - #l Val Phe Ala Leu Leu    #           750    - GAA GGA GAG CCT ATA GAA ATC TAC TAC TTT GA - #C AAA AAA ATT TTA CTT    2899    Glu Gly Glu Pro Ile Glu Ile Tyr Tyr Phe As - #p Lys Lys Ile Leu Leu    #       765    #                2917 AAG    Glu Lys Gly Glu Ile Lys        770    - TAGAAAGTCT CAAAAATTAA AGAAGTATGG AGCCATTGGC ACCATACTTC TT - #TAATTTTT    2977    - TTATATGTCG TTACTTGAAA GGGTGAGTGA CCCGCCTCCT ACACTTAAAT CA - #AAGTAATA    3037    - ATCTCCACTG CCTCGGAAGG AATATACTTT GATATAATAG GTTCCCGTTT GT - #GTTGGAAT    3097    - GTATGTTATT GTCTCCTGCC TTTGTGTTCC AGTTGAACTT TTGACAAGAG TA - #CCAGTTGG    3157    - GTCATAGAGG TATATATCGA AATCAGGGTT ATAGTTTGCC CAATCAGGTA TT - #ATGAAAGT    3217    - TATTGCAATA GGATATGATG TGTCTGTCAC ATTAAATGTC CAAATGTCAC TG - #TAACGAGA    3277    - CCCAGGAAGT GACTCTTTAG CATAATAGTG GTTTGGCGCA GATATATTAG TT - #CCTGTGAA    3337    #      3345    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:20 base pair - #s              (B) TYPE:nucleic acid              (C) STRANDEDNESS:double              (D) TOPOLOGY:linear    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:9:    #                 20GGT    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:20 base pair - #s              (B) TYPE:nucleic acid              (C) STRANDEDNESS:double              (D) TOPOLOGY:linear    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:10:    #20                ARCC    __________________________________________________________________________

What is claimed:
 1. An isolated DNA which encodes a trehalosephosphorylase which is obtainable from a microorganism of the genusThermoanaerobium and which hydrolyzes trehalose in the presence of acompound selected from the group consisting of an inorganic phosphoricacid, a salt thereof, and a mixture thereof to form D-glucose and acompound selected from a group consisting of β-D-glucose- 1-phosphoricacid, a salt thereof, and a mixture thereof.
 2. The DNA of claim 1,which has a nucleotide sequence of SEQ ID NO:5 or a complementarysequence thereof.
 3. The DNA of claim 1, wherein one or more bases arereplaced with different bases with respect to the degeneracy of geneticcode without altering the amino acid sequence encoded by the DNA.
 4. TheDNA of claim 2, which has been introduced into a self-replicable vector.5. The DNA of claim 1, which has been introduced into a host cell.
 6. Aprocess for producing a trehalose phosphorylase, which comprisesculturing a microorganism that produces trehalose phosphorylase in anutrient culture medium to produce the trehalose phosphorylase, andcollecting the produced trehalose phosphorylase from the culture,wherein said microorganism is a transformant which is prepared byintroducing the DNA of claim 1 into a host cell.